Western Blot: Novus Biologicals

NB100-2220

LC3B Antibody - BSA Free

(51 Reviews)

(1861 Publications)

0.1 ml

NB100-2220

Lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM CQ for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Polyclonal Antibody (Catalog # NB100-2220) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog# HAF008). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.

<b>Genetic Strategies Validation. </b>LC3B was detected in immersion fixed CQ treated HeLa cells (left) but was not detected in LC3B knockout HeLa cells (right) using rabbit anti-human LC3B polyclonal antibody (Catalog #NB100-2220) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights(TM) 557-conjugated anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.

<b>Biological Strategies Validation. </b>LC3B-Antibody-Western-Blot-NB100-2220-img0073.jpg

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LC3 accumulation in muscle fibers from patients with PAD (row B - E). Normal fibers from a Non-PAD sample (row A). Scale bar = 100 uM. Image from verified customer review.

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Detection of LC3B in mouse ES cell lysates using Rabbit anti- [Catalog # NB100-2220]. LC3 detected at a molecular weight of approximately 17 kDa. Atg5-/- ES cells from Dr. Noboru Mizushima [Mizushima, N. et al. J. Cell Biol. 152 (2001)] Photo courtesy of Dr. Beth Levine, UT SW Medical Center.

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<b>Biological Strategies Validation. </b>Lysates of mouse NIH3T3 and rat PC-12 cell lines untreated (-) or treated (+) with CQ. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL rabbit anti-LC3B polyclonal Antibody (Catalog # NB100-2220, Novus Biologicals), followed by 1:2000 dilution of goat anti-rabbit IgG secondary antibody. LC3 detected at a molecular weight of approximately 15 kDa in treated NIH3T3 and PC-12 cells.

<b>Biological Strategies Validation. </b>Detection of LC3I and LC3II in mouse cochlea cell line SV-K1 using Rabbit anti- [Catalog # NB100-2220]. Cells were treated with CQ (1 uM), and As2O3 (1 uM) for 24 hrs. Image from verified customer review.

<b>Biological Strategies Validation. </b>Autophagy Investigation. (A) Representative WBs (Western Blots) of LC3B-I, LC3B-II and GAPDH loading control in DANs derived from 4 iPD patients and 4 healthy controls. (B) WBs from three independent experiments were quantified by densitometry and LC3B-II normalized to a loading control. Grouping of data by healthy vs iPD as well as iPD stratified for T/T and C/C genotype. (C) Ratio of LC3B-II (normalized to loading control) following bafilomycin treatment/untreated. Citation: Marrone L, Bus C, Schondorf D, Fitzgerald JC, Kubler M, Schmid B, et al. (2018) Generation of iPSCs carrying a common LRRK2 risk allele for in vitro modeling of idiopathic Parkinson's disease. PLoS ONE 13(3): e0192497. //doi.org/10.1371/journal.pone.0192497

<b>Biological Strategies Validation. </b>LC3B-Antibody-Western-Blot-NB100-2220-img0067.jpg

<b>Biological Strategies Validation. </b>LC3B-Antibody-Western-Blot-NB100-2220-img0071.jpg

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Staining of rat brain tissue section. Analysis using Rabbit anti-LC3B Antibody [Catalog # NB100-2220]. Image from verified customer review.

Human ovarian Cancer tissue stained using heat mediated antigen retrieval in pH 6.0 citrate buffer at 1:200 dilution. Image provided by verified customer review.

FFPE (Formalin-Fixed Paraffin-Embedded) tissue section of mouse brain using 1:200 dilution of Rabbit anti-LC3B antibody [Catalog # NB100-2220]. The specific signal of LC3 was detected using HRP-conjugated secondary antibody with DAB (3, 3 -diaminobenzidine) reagent, and nuclei of cells were counterstained using hematoxylin. This LC3B antibody generated a low to moderate levels of cytoplasmic staining in the glial cells. The neurons depicted a moderate to strong staining for LC3 in their cytoplasm.

Analysis using the Biotin conjugate of Rabbit anti-LC3B Antibody [Catalog # NB100-2220]. Staining of brain, cerebral cortex, neurons with cell processes.

Image shows a specific band for LC3B at a molecular weight of approximately 15 kDa in 0.5 mg/mL of Neuro2A lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

TP53 knockout triggers a pro-mitophagic response.PINK1 (a,b, N = 8), LC3-2/LC3-1 (a,c, N = 8), p62 (a,d, N = 12), TIM23 (a,e, N = 10), TOM20 (a,f, N = 9), HSP60 (a,g, N = 16), optineurin (a,h, N = 9) & NDP52 (a,i, N = 8) protein levels were analyzed in control (p53+/+) or TP53-deficient (p53−/−) HAP1 cells as described in the Methods. Bars represent the means ± SEM of 3-4 independent experiments performed in triplicate & are expressed as percent of control (p53+/+) cells. Actin expression is provided as a representative gel loading control in a. Statistical analyses were performed with GraphPad Prism software by using unpaired Student’s t-test. Significant differences are: ** p < 0.01, ***p < 0.001 & ****p < 0.0001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29352272), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - SsD treatment of HeLa cells increases lysosomal pH & induces TFEB nuclear translocation. a, b SsD (15 μM) led to an increase of lysosomal pH in HeLa cells as measured using Lysosensor DND-189 (a) or the quantitative ratiometric LysoSensor Yellow/Blue DND-160 (b). The graphs in (b) & (c) represent data from three independent experiments, & data are expressed as the means ± S.D., n = 3. The asterisk (*) symbols indicates p < 0.05 by t-test analysis. c HeLa cells were transfected with TFEB-GFP & treated with or without SsD (15 μM) or underwent starvation before confocal imaging. d HeLa cells were transfected with siGFP or siTFEB & treated with or without SsD (15 μM), & then LC3, p62, TFEB, or GAPDH immunoblot analyses were performed Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30820356), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Endogenous LC3 accumulates around lysosomes.Wild type MEFs were treated with LCL161 (5 µM) or birinapant (50 µM) or DMSO as a control for 6 h. Cells were fixed & stained with antibodies against LAMP2 (red channel) & LC3 (green channel). Cells were imaged by confocal microscopy Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29743550), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Autophagy is involved in EV-A71 replication in HeLa cells. a VP1 synthesis was dramatically reduced by BAF (50 or 10 nM) treatment in EV71-infected cells compared to cells without BAF treatment. b ATG5 knockdown markedly inhibited EV-A71 infection in HeLa cells. c SsD treatment failed to further inhibit AV-A71 infection in ATG5-knockdown HeLa cells. d Rapamycin treatment enhanced EV-A71 infection in HeLa cells. e SsD treatment markedly inhibited EV-A71 infection in HeLa cells treated with or without Torin-1 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30820356), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Topotecan treatment triggers autophagy in colon cancer cells.A. HCT116, LS-174T & HT29 cells were transfected for 24 h with an expression construct encoding LC3 fused to the yellow fluorescent protein (YFP-LC3). Thereafter, the cells were treated with or without 1 µg/mL topotecan (TPT) for 24 h & visualised under a confocal microscope. B. The indicated cells were treated with the indicated concentrations of topotecan for 24 h. The lysates were analysed by immunoblotting with The LC3 & P62 antibodies. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23024792), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - LC3 expression in the adjacent non-tumor tissues by immunohistochemistry stainingRepresentative images of areas according to the proportion of positive cells (A–D) & intensity of staining (E–H). (A) none, (B) < 10%, (C) 10–50%, (D) > 50%; & staining (E) absent, (F) weak, (G) moderate, (H) strong. (upper panel, x200; lower panel, x400). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29190884), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - P53 mediates topotecan-induced autophagy through the activation of sestrin 2 & AMPK in colon cancer cells with wild-type p53.A. HCT116 & LS-174T cells were treated with various concentrations of TPT for 24 h. The levels of P53, sestrin2 & p-AMPK were analysed by immunoblotting. B. HCT116 cells were transfected with p53 or sestrin 2 siRNAs for 24 h, treated with or without 1 µg/mL TPT for an additional 24 h, & the indicated proteins were then analysed by immunoblotting. C. HCT116 cells were transfected with LKB1 or CaMKK beta siRNAs, treated with or without 1 µg/mL TPT for an additional 24 h, & the indicated proteins were detected by immunoblotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23024792), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Specific inhibitor SP600125 or siRNA directed JNK blocked ceramide-induced autophagy & up-regulation of LC3 expression. (A) SUNE1 cells were treated with 20 μM ceramide for 24 h in the absence or presence of SP600125 or JNK1/2 siRNA. Lysates were analyzed by immunoblotting. (B) Autophagosome formation was visualized using YFP-LC3 expressing & observed under a confocal microscope. Representative immunofluorescence pictures are shown at the original magnification × 1000. (C) The expression of LC3 mRNA was examined by RT-PCR analysis. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21943220), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Glutamine deprivation induces IL-8 secretion.(a) RT–PCR analysis of LC3, IL-8, CHOP & GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). (b) Immunoblot analysis of ER stress- & autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate. (c) Cytokine array analysis of conditioned media from U2OS & A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. (d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS & A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms5900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - NDs API improves ATO-based therapy in vivo. HepG2 tumor-bearing nude mice administered intravenously (i.v.) w/ NS (200 μL), NDs (5 mg kg−1), ATO (0.75 mg kg−1), ATO (1.5 mg kg−1), NDs–0.75 mg kg−1 ATO mixture,/NDs–1.5 mg kg−1 ATO mixture daily for 5 consecutive days every week, w/ 13-week cycles c Immunoblots of autophagy-related proteins LC3-II, p62 (left); semi-quantified analysis (n = 3) in tumor tissues from mice after treatment (right). GAPDH used as loading control. Normalized band densities shown below each band. Image collected & cropped by CiteAb from following publication (//pubmed.ncbi.nlm.nih.gov/30341298), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - NDs API improves the ATO-based therapy in vivo. HepG2 tumor-bearing nude mice administered intravenously (i.v.) w/ NS (200 μL), NDs (5 mg kg−1), ATO (0.75 mg kg−1), ATO (1.5 mg kg−1), NDs–0.75 mg kg−1 ATO mixture,/NDs–1.5 mg kg−1 ATO mixture daily for 5 consecutive days every week, w/ 13-week cycles. g Immunostaining of LC3 & ATG13 in tumor tissues of NDs–1.5 mg kg−1 ATO-treated mice. Schematic showing (left) & imaging (right) indicate that LC3/Atg13 puncta in periphery & around the blood vessels inside the tumors. Scale bars: 20 μm. Error bars s.d. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30341298), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - LC3, CRF, & HIF-1 alpha expression. (a) for each antibody the spatial distribution of immunohistochemical staining was the same in cases with spontaneous & induced labor. (b) The analysis of data from optical density values of Western blotting bands shows no differences of LC3-II & HIF-1 alpha expression between groups. On the contrary CRF is higher in spontaneous labor. LC3-II, HIF-1 alpha , & CRF are normalized onto alpha -tubulin. (c) No correlation between LC3-II & CRF expression was found. SP: spontaneous labor; PG: induction with prostaglandin; PO: induction with prostaglandin & oxytocin; OX: induction with oxytocin. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23956998), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - The time-dependent effects of melatonin on the LC3-II & Beclin 1 expression in U87MG (a, b) & A172 (c, d) cells. The cells were treated with 1 mM melatonin for 4, 12, 24 or 36 h. PBS was used as a negative control, & rapamycin (Rap 200 nM) served as a positive control. The levels of LC3-II & Beclin-1 expression were determined by a Western blot analysis. The protein bands for each regimen were quantified by densitometry, & their differences are presented in the graph. The values represent the mean ± S.E.M. for three separate determinations. Statistical significance * at P < 0.05 or ** at P < 0.01 compared with untreated controls Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31870319), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Melatonin-induced autophagy was abolished by treatment with an autophagy inhibitor, 3-MA. The results of the Western blotting of LC3 & Beclin-1 in the U87MG (a, b) & A172 (c, d) cells treated with PBS (control) or melatonin (MLT 1 mM) for 24 h in the presence & absence of 3-MA (10 mM). Statistical significance * at P < 0.05 compared with untreated controls or the corresponding group Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31870319), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - TonEBP haploinsufficient mice do not show altered autophagy in NP. LC3 immunofluorescence staining of intervertebral discs from 4-month-old TonEBP+/+ (a–c) & haploinsufficient TonEBP+/− (d–f) mice demonstrated similar pattern & distribution of LC3 positive autophagosomes. (a,d) White dotted line demarks the NP tissue compartment. (c,f) Magnified images of dotted inserts from B & E respectively. White arrows indicate LC3-positive autophagosomes. Scale bar: 200 μm for (a) & (d); 20 μm for (b), (c), (e), & (f). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28674405), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - PLD1 silencing prevents TNP-induced autophagy & decreases in HIV p24 release. Mock- & HIV-infected macrophages (A to C) & CD4+ T cells (D to F) transfected with PLD1 siRNA (siPLD1) or scrambled siRNA (siNS) were exposed to vehicle, 400 μg ml−1 RBC-NP (R), or 400 μg ml−1 TNP (T) for 4 h, washed three times with PBS, & then incubated for a further 24 h. n = 4. (A & D) (Bottom) Representative western blots of PLD1. (Top) Densitometric analysis of blots. (B & E) (Top) Representative western blots of LC3B isoforms & SQSTM1. (Bottom) Densitometric analysis of blots. (C & F) Enzyme-linked immunosorbent assays were performed for HIV p24 antigen in the supernatant. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32934078), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - GII-NTPase possesses proapoptotic activity, which can be further enhanced by Nterm or P22. (A) Effects of GII-NTPase or its deletion mutants on cell apoptosis, autophagy, & ER stress. After transfection of the indicated GII-NTPase expression plasmids into 293T cells for 24 h, the expression of specific markers for apoptosis (PARP or caspase 3), autophagy (LC3B), & ER stress (GRP78) were examined by Western blotting. (B) Enhancement of the GII-NTPase-mediated apoptosis by Nterm or P22. 293T cells were cotransfected with the indicated plasmids expressing GII-NTPase, Nterm, or P22. After 24 h of transfection, the expression of cleaved PARP & caspase 3 in the cells was analyzed by Western blotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29212938), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - The concentration-dependent effects of melatonin on the LC3-II & Beclin-1 expression in U87MG (a, b) & A172 (c, d) cells. The cells were treated with 100, 200, 1000 or 2000 μM of melatonin for 24 h. PBS was used as a negative control, & rapamycin (Rap 200 nM) served as a positive control. The levels of LC3-II & Beclin-1 expression were determined by a Western blot analysis. The protein bands for each regimen were quantified by densitometry, & their differences are presented in the graph. The values are represented as the mean ± S.E.M. for three separate determinations. Statistical significance * at P < 0.05 or ** at P < 0.01 compared with untreated controls Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31870319), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: LC3B Antibody - BSA Free [NB100-2220] - Western blot analysis of human recombinant microtubule-associated proteins 1A/1B light chain 3A & 3B probed with either a the Abgent anti-LC3 antibody (AP1802a), or b MBL anti-LC3A/B antibody. LC3 bands were quantified using the Odyssey Infrared Imaging System (Li-Cor), & data presented as integrated intensities (n = 3). c Analysis of LC3B distribution in untreated OE21 & OE33 esophageal cancer cell lines. Arrows indicate diffuse cytoplasmic LC3B expression (magnification 400×) (n = 6). d Analysis of LC3B distribution in untreated KYSE450 & OE19 esophageal cell lines. Arrows indicate the presence of LC3B crescent or ring-like (upper panels) & globular (lower panels) structures in KYSE450 & OE19 cells (magnification 1000×) (n = 6) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26265176), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Expression of autophagy-related proteins. MDA-MB231 & MCF-7 cells were treated with different concentrations of T33 for 48 h & a expressions of LC3-I & LC3-II proteins in MDA-MB231 & MCF-7 cells were detected with immunoblotting. Ratios of LC3-II/ beta -Actin in b MDA-MB231 & MCF-7 cells are shown. Similar results were observed in triplicate experiments. The superscripts 1, 2, & 3 refer to significant differences (P < 0.05) from the MDA-MB231 control, MCF-7 control & MDA-MB231, respectively Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31409331), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - CB1 knockout elevated autophagy in the liver of HBs transgenic mice. a Representative immunohistochemical analysis of paraffin-embedded liver sections from 52-week-old mice was performed using anti-LC3B antibody. Original image magnification ×1000, bar 40 µm. b Quantification of LC3B puncta assessed with ImageJ software & expressed as % of LC3B staining/field. c Western blot analysis of lysates from 52-week-old mice was performed using specific anti-LC3B & anti-p62 antibodies. d Representative immunofluorescence analysis of paraffin-embedded liver sections of 52-week-old mice was performed using specific anti-LAMP1 antibodies (red). Nuclei were stained with DAPI (blue). Magnification ×1000, bar 40 µm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31570772), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - TLR4 is critical to Hsp70/Hsp90-induced muscle wasting in mice. a Hsp70 & Hsp90-induced muscle catabolism in mice is dependent on TLR4. Wild-type & TLR4 null mice were injected with rHsp70 & rHsp90 & analyzed for catabolic response in TA as described in Fig. 4. b Hsp70 & Hsp90-induced muscle wasting is TLR4-dependent. Wild-type & TLR4 null mice injected with rHsp70 & rHsp90 are further analyzed for muscle wasting. Scale bar, 100 μm. Data (n = 5) were analyzed by analysis of variance or χ2 analysis (for CSA). * denotes a difference (P < 0.05) Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-017-00726-x), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - Representative images of gastrocnemius muscle sections from non-PAD & PAD subjects stained for LC3, an autophagosome marker (red) & LAMP2, a lysosome marker (green). A In non-PAD, very little LC3 & LAMP2 staining is apparent. In PAD, rare fibers show co-localization of accumulated LC3 & LAMP2 (B), whereas the majority of fibers have elevated LC3 accumulation but no co-localization with LAMP2 (C). Arrows in the same column point to the same areas of LC3 accumulation in the center of the fiber with or without LAMP2 co-localization. Scale bar = 50μm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27687713), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Vacuolin-1 analogues identified via virtual screening induced the accumulation of both LC3B-II & SQSTM1 in HeLa cells in a dose dependent manner after a 6 h treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28555021), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Inhibition of autophagy in MYOCD-knockdown cells restored the expression of contractile proteins.HA-VSMCs were transfected with shMYOCD plasmid for 4 h & then treated with 5 mM 3-MA for another 48 h. Western blot images & quantification in each group were shown (*P < 0.05, **P < 0.01, n = 3). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35136037), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - Accumulation of an autophagosome marker in the presynaptic terminals of NMJs of SOD1G93A mice (A) Representative confocal micrographs of immunohistochemistry showing accumulation of the autophagosome marker LC3 (grayscale in left panels, green in right panels) in presynaptic terminals of diaphragm NMJs in SOD1G93A mice at P120, but not at NMJs of wild-type mice at P140. Nerves were stained using anti-neurofilament & anti-SV2 antibodies (blue), & postsynaptic acetylcholine receptors were labeled with Alexa 594-conjugated alpha -bungarotoxin (red). In these cross-section images of NMJs, presynaptic terminals are above the bungarotoxin signal, & postsynaptic myotubes are below the bungarotoxin signal. Scale bar: 10 μm. (B) The number of NMJs with LC3 puncta was significantly higher in SOD1G93A mice than in wild-type mice. Quantifications are from n = three animals & 268–305 NMJs each genotype in confocal images. Asterisks indicate a significant difference by un-paired t-test (**p < 0.01). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28890682), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Autophagy is dynamically regulated by migration-promoting & migration-inhibiting cues & is required for the recycling of paxillin.(A–C:) Immunoblotting for the lipidated form of LC3 (LC3-II), p62, & paxillin on RMS samples dissected from acute sections previously incubated with BDNF, GABA, GM60001, Y27632, or blebbistatin for 2 hr. GAPDH was used as a housekeeping protein. (D–E:) Quantification of LC3-II & paxillin levels after the pharmacological manipulation of cell migration (n = 5–7 mice for both groups, *p<0.05 & **p<0.005 with a Student t-test). (F): Example of a cell infected with a retrovirus expressing the LC3-GFP-RFP fusion protein & immunostained for Lamp1 to label autophagosomes (GFP+/RFP+) & autolysosomes (RFP+/Lamp1+). (G:) Percentage of autophagosomes & autolysosomes after a 2 hr incubation with BDNF, GABA, GM60001, Y27632, or blebbistatin. The autophagosome/autolysosome ratio was assessed for each cell, & the results are expressed as means ± SEM. ***p<0.001 with a one-way ANOVA followed by a post hoc LSD-Fisher test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32985978), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - OSI induces autophagy through upregulation of MCT1 in CRC cells.a Immunoblotting analysis of MCT1 expression in CRC cells treated with or without 5 μM OSI for 24 h. b Immunoblotting analysis of MCT1 & phosphorylated AMPK in tumor xenografts obtained from vehicle- or OSI-treated mice. (Each protein of interest from each group was electrophoretically transferred onto a PVDF membrane, incubated with indicated primary & secondary antibodies, & developed as a digital image.) c Immunohistochemical analysis of MCT1 expression in tumor xenografts. Scale bar, 50 μm. d Relative intensity of MCT1 staining in (c). e CRC cells were transfected with siScramble or siMCT1 for 24 h, followed by treatment with or without 5 μM OSI for another 24 h. The protein levels of LC3, phosphorylated AMPK, phosphorylated LKB1 & MCT1 were analyzed by immunoblotting. f CRC cells were treated as in (e), the endogenous LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. g The number of LC3 puncta in (f). h CRC cells were transfected with empty vector or Flag-MCT1 plasmid for 48 h, the protein levels of MCT1 & phosphorylated AMPK were analyzed by immunoblotting. i CRC cells were treated as in (h), the endogenous LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. j The number of LC3 puncta in (i). k Immunoblotting analysis of LC3, MCT1 & phosphorylated AMPK levels in CRC cells co-transfected with Flag-MCT1 & DN-AMPK plasmids for 48 h. l CRC cells were treated as in (k), the endogenous LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. m The number of LC3 puncta per cell in (l). Data are presented as mean SEM, Student’s t-test, & are representative of three independent experiments. *P < 0.05; ***P < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31409796), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Screening of potent NAPIs in HepG2. a Immunoblots for autophagy-related proteins LC3-II, p62 (left); semi-quantified analysis (n = 3) in various nanoparticles treated cells (right). CQ & Rapamycin (Rapa) were used as positive controls for autophagy inhibition & autophagy activation, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Normalized band densities were shown below each band. b Fluorescence images of mCherry-GFP-LC3 cells after incubation with CQ or NDs for 48 h (autophagosomes: mCherry+/GFP+ yellow puncta; autolysosomes: mCherry+/GFP) & quantification of the number of LC3 puncta per cell in cells (10 cells per group). Scale bar: 10 μm. c Immunoblots for autophagy-related proteins LC3-II, p62 (left); semi-quantified analysis (n = 3) in CQ, NDs, or CQ–NDs-treated cells (right). &P < 0.05, significantly different from NDs. GAPDH was used as the loading control. Normalized band densities were shown below each band. d Left: Cell viability after incubation with ATO or various NAPIs–ATO mixture for 48 h (n = 3). ##P < 0.01 by t-test, significantly different from ATO. Right: Cell viability after 48 h NDs–ATO treatment with RNAi of autophagy proteins ATG5 & ATG7 (n = 3). e, f Immunoblots for autophagy-related protein LC3-II & autolysosomal process-related protein NUPR1, SNAP25, VAMP8 in NDs-treated cells. g Immunoblots for autolysosomal process-related protein NUPR1 after NDs treatment with RNAi of autophagy proteins ATG5 & ATG7. GAPDH was used as the loading control. Error bars are s.d. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30341298), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Glutamine deprivation induces autophagic flux.(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) & 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student’s t-test. *P<0.05. (c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before & during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 & LC3-II proteins. (d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm. (f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms5900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Glucocorticoids promote BCG survival in macrophages by inhibiting autophagy. (a) RAW264.7 cells were pretreated with dexamethasone (DEX) (1 μM) or vehicle control ethanol (mock) for 6 hr & then challenged with M. bovis BCG (MOI 5) or treated with the vehicle control DMSO or rapamycin (4 μM). LC3 level was detected with Western blot analysis. & GAPDH was used as the loading control. Full-length blots are presented in Supplementary Fig. 12. Data are representative of three independent experiments with similar results. (b,c) RAW264.7 cells were treated as described in (a), & viable bacilli were determined by CFU assays at 6 hpi (b). Survival rate was calculated compared with that of mock-treated cells (c). Data are shown as the mean ± SEM of three independent experiments. *p < 0.05. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-01174-9), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - CysC regulates AMPK activity during the induction of autophagy. (a) Immunoblotting analysis of autophagy regulators. N2a cells expressing SOD1 treated with or without CysC (1 μM) for 6 h. The lysates analyzed by immunoblotting using antibodies for phosphorylated AMPK (pAMPK), phosphorylated mTOR (pmTOR), phosphorylated PKCδ (pPKCδ), Myc & beta -actin. (b) Inactivation of AMPK by mutant SOD1 expression. Each relative pAMPK level normalized by beta -actin in (a) is quantified. *P<0.05, **P<0.01 versus mock. (c) Activation of AMPK by CysC treatment. Relative levels of pAMPK for CysC-treated samples normalized by that of PBS-treated control, which is shown as the broken line, in (a) quantified. *P<0.05 versus PBS-treated controls. (d) CysC induced autophagy through the AMPK activation. N2a cells treated with CysC (1 μM), CC (5 μM) or AICA-riboside (AICAR, 5 mM) for 12 h. The lysates analyzed by immunoblotting using antibodies against pAMPK, LC3 & Tubulin (left panel). Quantification of immunoblots was plotted (right panel). (e) The effect of pAMPK activation on neuroprotection by CysC. N2a cells expressing G85R SOD1 mutant treated with CysC (0.2 μM), CC (5 μM) or AICAR (5 mM). Cell viability was measured by the MTS assay. Data expressed as means±S.E.M. from 3 independent experiments. Each experiment was performed in triplicate. *P<0.01 compared to non-treated control, #P<0.01 compared to CysC-treated one. (f) Immunoblotting analysis of pAMPK in SOD1 transgenic mouse spinal cords. The spinal cord lysates from the transgenic mice at indicated ages analyzed for the levels of pAMPK, SOD1 & beta -actin (upper panel). Expression levels of pAMPK normalized by beta -actin (lower panel). All data expressed as means±S.E.M. from 3 independent experiments Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2014459), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Vps34 inhibition blocks autophagy initiation & causes accumulation of ubiquitin-positive & p62-positive structures. a Representative confocal images of cortical neurons treated with vehicle, Bafilomycin A1 (BafA1) at 50 nM, VPS34IN1 at 3 µM or cotreated for 3 h. Arrows highlight LC3 & p62 structures. Right panel, bar graphs denote average object intensity, per cell (mean ± SEM, N = 49-60 cells, from three independent experiments). Scale bar, 10 µm. ***p < 0.001 in one-way ANOVA, Holm–Sidak’s multiple comparisons test. b Representative confocal images of cortical neurons treated as in a & immunostained for LAMP-1, LC3, & p62. Airyscan insets highlight position of LC3 & p62 structures relative to LAMP-1-positive membranes. Scale bar, 10 µm. c Representative confocal images of cultured cortical neurons treated with vehicle or VPS34IN1 at 3 µM for 24 h. Arrows highlight p62 & ubiquitin colocalization. Scale bar, 10 µm Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-017-02533-w), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - ORP1L & VAP-A form cholesterol-dependent ER-AV contact sites(a) MelJuSo cells expressing GFP or GFP-tagged ORP1L mutants were fixed & stained for LC3 & Calnexin. Y477 & D478 in the FFAT motif were mutated to alanines (A) in the ORP1L ydaa mutant. Scale bar, 10 μm. Right: co-immunoprecipitation for ORP1L (mutants) with VAP-A. GFP-ORP1L mutants or GFP were isolated from lysates of HEK293T cells co-overexpressing HA-VAPA using GFP-Trap beads. Western blot filters were probed for isolated GFP-tagged proteins, the associated HA-VAP-A & the input HA-VAP-A, as indicated. (b) Cryo-immuno-EM on HeLa cells expressing HA-LC3 & GFP-ORP1L delta ORD, as detected by HA10 nm & GFP15 nm gold antibodies. Insets show ORP1L labelling in the membrane contact site between ER & autophagosome. The membranes of the ER are depicted in the bottom inset. Scale bar, 50 nm. (c) Three-colour super-resolution image of an autophagosomal vesicle labelled by LC3 (green), ORP1L (blue) & the ER protein VAP-A (red). Scale bar, 500 nm (d) MelJuSo cells cultured either in lipid depleted serum or control medium were fixed & stained for LC3 & ER marker Calnexin. Scale bar, 10 μm. Right panel: Manders coefficient for LC3 localization to the ER was calculated on at least 10 cells over three independent experiments. Bars indicate mean+s.d. Student's t-test statistical analysis (****P<0.0001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27283760), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Abrogation of autophagy by siRNAs targeting autophagy-related genes in gastric cancer cells induced tumor-intrinsic PD-L1 expression. a Inhibition of autophagy by knockdown of ATG5 in AGS & NCI-n87 gastric cancer cells induced the expression of PD-L1 in the presence & absence of INF-gamma (200 U/mL) as shown by flow cytometry analysis at 48 h post-transfection. b The induction of PD-L1 was confirmed by Western blots at 72 h post-transfection. The knockdown efficacies of ATG5 & ATG7 siRNA were verified. The conversion of LC3B-I to LC3B-II was reduced. Results were averaged & blots were representative of 4 independent experiments. The ratio of PD-L1 MFI minus isotype control was shown as mean ± S.D. relative to Ctrl from 4 independent experiments, *p < 0.05 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30925913), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - OSI induces autophagy through upregulation of MCT1 in CRC cells.a Immunoblotting analysis of MCT1 expression in CRC cells treated with or without 5 μM OSI for 24 h. b Immunoblotting analysis of MCT1 & phosphorylated AMPK in tumor xenografts obtained from vehicle- or OSI-treated mice. (Each protein of interest from each group was electrophoretically transferred onto a PVDF membrane, incubated with indicated primary & secondary antibodies, & developed as a digital image.) c Immunohistochemical analysis of MCT1 expression in tumor xenografts. Scale bar, 50 μm. d Relative intensity of MCT1 staining in (c). e CRC cells were transfected with siScramble or siMCT1 for 24 h, followed by treatment with or without 5 μM OSI for another 24 h. The protein levels of LC3, phosphorylated AMPK, phosphorylated LKB1 & MCT1 were analyzed by immunoblotting. f CRC cells were treated as in (e), the endogenous LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. g The number of LC3 puncta in (f). h CRC cells were transfected with empty vector or Flag-MCT1 plasmid for 48 h, the protein levels of MCT1 & phosphorylated AMPK were analyzed by immunoblotting. i CRC cells were treated as in (h), the endogenous LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. j The number of LC3 puncta in (i). k Immunoblotting analysis of LC3, MCT1 & phosphorylated AMPK levels in CRC cells co-transfected with Flag-MCT1 & DN-AMPK plasmids for 48 h. l CRC cells were treated as in (k), the endogenous LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. m The number of LC3 puncta per cell in (l). Data are presented as mean SEM, Student’s t-test, & are representative of three independent experiments. *P < 0.05; ***P < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31409796), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Screening of potent NAPIs in HepG2. a Immunoblots for autophagy-related proteins LC3-II, p62 (left); semi-quantified analysis (n = 3) in various nanoparticles treated cells (right). CQ & Rapamycin (Rapa) were used as positive controls for autophagy inhibition & autophagy activation, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Normalized band densities were shown below each band. b Fluorescence images of mCherry-GFP-LC3 cells after incubation with CQ or NDs for 48 h (autophagosomes: mCherry+/GFP+ yellow puncta; autolysosomes: mCherry+/GFP) & quantification of the number of LC3 puncta per cell in cells (10 cells per group). Scale bar: 10 μm. c Immunoblots for autophagy-related proteins LC3-II, p62 (left); semi-quantified analysis (n = 3) in CQ, NDs, or CQ–NDs-treated cells (right). &P < 0.05, significantly different from NDs. GAPDH was used as the loading control. Normalized band densities were shown below each band. d Left: Cell viability after incubation with ATO or various NAPIs–ATO mixture for 48 h (n = 3). ##P < 0.01 by t-test, significantly different from ATO. Right: Cell viability after 48 h NDs–ATO treatment with RNAi of autophagy proteins ATG5 & ATG7 (n = 3). e, f Immunoblots for autophagy-related protein LC3-II & autolysosomal process-related protein NUPR1, SNAP25, VAMP8 in NDs-treated cells. g Immunoblots for autolysosomal process-related protein NUPR1 after NDs treatment with RNAi of autophagy proteins ATG5 & ATG7. GAPDH was used as the loading control. Error bars are s.d. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30341298), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - OSI induces autophagy in CRC cells in vitro & in vivo.a Immunoblotting analysis of LC3, Atg5, & p62/SQSTM1 expression in CRC cells treated with indicated concentrations of OSI for 24 h. b The formation of endogenous LC3 puncta in cells treated with DMSO or 5 μM OSI for 24 h. c Total number of endogenous LC3 puncta per cell in (b). d, e LC3 expression in xenograft tissues was examined by IHC. Representative images were provided as indicated in (d) & relative intensity of LC3 staining was quantified in (e). f Immunoblotting analysis of LC3 & p62/SQSTM1 expression in tumor xenografts (Each protein of interest from each group was electrophoretically transferred onto a PVDF membrane, incubated with indicated primary & secondary antibodies, & developed as a digital image.) g Relative intensity of LC3 in (f). h Co-immunoprecipitation analysis of the interaction between Beclin 1 & Bcl-2 in CRC cells treated with or without 5 μM OSI for 24 h. i Immunoblotting analysis of LC3 expression in CRC cells treated with or without 5 μM OSI in the presence or absence of 5 mM 3-MA for 24 h. j CRC cells were treated as in (i), the LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. k, l Immunoblotting analysis of LC3 expression in CRC cells transfected with siScramble, siATG5 (k), or siBECN1 (l) for 24 h, followed by treatment with or without 5 μM OSI for another 24 h. m CRC cells were treated as in (k, l). The LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. Data are presented as mean SEM, Student’s t-test, & are representative of 3 independent experiments. **P < 0.01; ***P < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31409796), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - OSI induces autophagy in CRC cells in vitro & in vivo.a Immunoblotting analysis of LC3, Atg5, & p62/SQSTM1 expression in CRC cells treated with indicated concentrations of OSI for 24 h. b The formation of endogenous LC3 puncta in cells treated with DMSO or 5 μM OSI for 24 h. c Total number of endogenous LC3 puncta per cell in (b). d, e LC3 expression in xenograft tissues was examined by IHC. Representative images were provided as indicated in (d) & relative intensity of LC3 staining was quantified in (e). f Immunoblotting analysis of LC3 & p62/SQSTM1 expression in tumor xenografts (Each protein of interest from each group was electrophoretically transferred onto a PVDF membrane, incubated with indicated primary & secondary antibodies, & developed as a digital image.) g Relative intensity of LC3 in (f). h Co-immunoprecipitation analysis of the interaction between Beclin 1 & Bcl-2 in CRC cells treated with or without 5 μM OSI for 24 h. i Immunoblotting analysis of LC3 expression in CRC cells treated with or without 5 μM OSI in the presence or absence of 5 mM 3-MA for 24 h. j CRC cells were treated as in (i), the LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. k, l Immunoblotting analysis of LC3 expression in CRC cells transfected with siScramble, siATG5 (k), or siBECN1 (l) for 24 h, followed by treatment with or without 5 μM OSI for another 24 h. m CRC cells were treated as in (k, l). The LC3 puncta were analyzed by immunofluorescence. Scale bar, 10 μm. Data are presented as mean SEM, Student’s t-test, & are representative of 3 independent experiments. **P < 0.01; ***P < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31409796), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Growth retardation & shortened lifespan of the mice with deletion of an autophagy-essential gene Atg7 in hematopoietic system. (A) Three genotypes for wild-type, heterozygote, & homozygote for Atg 7 deletion in hematopoietic system with representative images of the mice. The images were taken at age of 10 weeks. (B) PCR Genotyping analysis of the offsprings from Atg7f/f mice crossing Vav-iCre mice to screen Atg7f/f;Vav-iCre mice. The sequences for the primers used in PCR are given in the method section, & their PCR amplified bands representing specific genotypes were indicated in the agarose gel electrophoresis films. (C) Examination of Atg7 expression in wild-type & the Atg7-deleted mice. Upper panel, quantitative PCR analysis of Atg7 transcription normalized to Gapdh transcript in different organs; lower panel, western blotting analysis of autophagy-essential protein ATG7 & lipidation of LC3 in different organs. GAPDH used as a loading control. (D) Growth comparison between wild-type & Atg7-deleted mice. Wild-type mice progressively gain weight before age of 60 weeks (left panel), but Atg7-deleted mice cease weight gain at about age of 6 weeks (right panel). (E) Measurement of lifespan of wild-type & Atg7-deleted mice. (F) Immunohistological examination of heart, liver, lung & thymus from 10 weeks old wild-type & Atg7-deleted mice by HE staining. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31327762), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Overexpression of wild-type MAGEA6, but not mutant MAGEA6, suppresses autophagy in PDAC cell lines.(A) Immunoblot analysis of autophagy signaling in HPDE-iKRAS cells expressing GFP (Vec) & MAGEA6 variants. (B) Immunofluorescence staining of LC3B puncta in the transduced HPDE-iKRAS cells. Representative photos (left) & statistical analysis (mean ± standard deviation of counted cells, N=~100 per cohort) are shown. *p=0.002; two-tailed unpaired t-test. (C) Immunoblot analysis of autophagy substrate SQSTM1/p62 in the transduced HPDE-iKRAS cells treated with BafA1 for the indicated time points., Immunoblot analysis of (D) autophagy signaling & (E) SQSTM1/p62 accumulation in wild-type MAGEA6 expressing & (F) autophagy signaling in MAGEA6H305fs* expressing HPDE-iKRAS cells under nutrient-deficient conditions. (G) Immunoblot analysis of autophagy signaling in wild-type MAGEA6 expressing cells under prolonged nutrient-deficient conditions.Immunoblot analysis of autophagy signaling of transduced AsPC-1 & MIA PaCa-2.Immunoblot analysis of the accumulation of autophagy substrate SQSTM/p62 in the transduced HPDE-iKRAS cells under BafA1 for the indicated time points.Immunoblot analysis of autophagy activity in transduced HPDE-iKRAS cells under nutrient-depleted conditions as indicated. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32270762), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Inhibition of LRRK2 alters the autophagy/lysosomal pathway in H4 neuroglioma cells. A) LC3-II levels increase upon LRRK2-in1 inhibitor treatment (1 μM, overnight treatment; quantification from 3 independent replicates, the plot shows mean & SD, * indicates significance (p < 0.05)). B) Dose dependent increase in LC3-II upon overnight treatment with LRRK2-in1. C) MTT assay showing no alteration of cell viability upon over night treatment with LRRK2-in1 from 1 to 5 μM. A small toxic effect appeared with the higher dose (10 μM). The plot shows mean & SD, * indicates significance (p < 0.05). D) LRRK2 knockdown cells display reduced response to LRRK2-in1. LRRK2 protein levels are decreased in shRNA stable line compared to wild type cells (right panel), & knockdown of LRRK2 reduces response to 1 μM LRRK2-in1 treatment compared to wild type or scrambled shRNA cells (left panel). E) Western blot analysis of H4 cells treated with DMSO & LRRK2-in1 (5 μM, 2.5 hours treatment) in the presence & absence of 40 nM bafilomycin added at the same time as the inhibitor. Quantification of three replicates is shown in the right hand panel, the plot shows mean & SD, * indicates significance (p < 0.05). F) LRRK2-in1 increases LC3-II levels independent of mTORC1 activity. P70S6K & phoshoThr389-P70S6K; S6 & phosphoSer235/236-S6 levels are shown in control, starvation & amino-acid stimulated conditions. LRRK2-in1 (1 μM over night) treatment does not alter phosphorylation of P70S6K & S6 in conditions that show increased levels of LC3-II. G) Neutral red staining. The plot shows mean & SEM. * indicates significance (p < 0.05). Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S0167488913002954), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - SQSTM1 recruited ARHGAP5-AS1 for autophagic degradation.a ARHGAP5-AS1 expression in resistant cells after transfecting siNC or SQSTM1 siRNAs was assessed using qRT-PCR. **p < 0.01. b ARHGAP5-AS1 expression in sensitive cells after SQSTM1 overexpression was detected by qRT-PCR. c The interaction of ARHGAP5-AS1 with SQSTM1 in SGC-R cells was analyzed by RIP assay followed with qRT-PCR. IgG was served as the negative control. d RNA pull down assay was performed to verify the binding of SQSTM1 to ARHGAP5-AS1 in SGC7901 or SGC-R cells. beta -actin was served as negative control. NC: no probe. The interaction of ARHGAP5-AS1 with SQSTM1 in SGC-R cells before & after EBSS treatment (4 h) were analyzed using RIP assay (e) & RNA pull down assay (f). g The interaction of various SQSTM1 constructs with ARHGAP5-AS1 were analyzed using RIP assay. h The interaction of various ARHGAP5-AS1 fragments with SQSTM1 was analyzed by RNA pull down assay. i Half-life of ARHGAP5-AS1 in SGC7901 & SGC-R cells with different SQSTM1 expression status were determined by qRT-PCR. The colocalization of ARHGAP5-AS with SQSTM1 (j) & LC3B (k) in SGC-R cells treated as indicated were analyzed by combined FISH & IFC assay (original magnification, ×100). Scale bar: 100 μm (j) & 10 μm (k) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31097692), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Effects of autophagy & caspase inhibitors on survival of neurons after glucose reperfusion (GR).After 3 h GD, neuro2a cells were replaced by 20-Methyladenine (3MA); Bafilomycin A1 (BA1) & z-VAD-FMK (z-VAD) were added at final concentrations of 5 mM, 5 nM & 20 uM, respectively. (A) Cell viability was assessed using the MTT assay after treatment of autophagy & pan-caspase inhibitors. MTT mitochondrial reduction was shown as a relative percentage of the MTT values at the indicated time point (3 h GD/48 h GR) from three independent experiments ± SD (*p<0.05, **p<0.001). (B) Changes in protein levels by inhibitors of autophagy or caspase activation were analyzed by immunoblot. Proteins were blotted with anti-LC3, p62, cleaved-caspase 3 & PARP antibodies. Tubulin was used as loading control. (C) Quantification of immunoblot data in (B). The amount of each protein was normalized against the amount of tubulin. Data represent the mean ± SD (*p<0.05, n = 3). (D) Cells transiently expressing GFP-LC3 (green) were treated with 3MA, BA1 or z-VAD for 48 h of GR after 3h GD. Nuclei were stained with DAPI (blue). Scale bar represents 5 µm. Quantification shown on the right graph represents mean GFP-LC3 puncta per cell (n = 10 cells per condition) from three independent experiments ± SD. The asterisks (*) indicate significant differences in the values.*p<0.05, **p<0.001. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0076466), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Exposure of microglia to cART resulted in blockade of autophagosome–lysosome fusion. (A) rPMs were seeded into a 12-well plate followed by tandem fluorescent-tagged MAP1LC3B plasmid. Next, cells were exposed to cART (5 µM each of TDF, FTC, & DTG) for an additional 24 h & observed by confocal imaging. The results showed that cART exposure significantly increased the formation of autophagosomes (yellow puncta). (B) Representative bar graph showing the number of autophagosome (yellow puncta) per cell. (C) Representative bar graph showing the number of autolysosome (red puncta) per cell. (D) rPMs were seeded into 12-well plates followed with cART exposure for 24 h. Cells were then double immunostained with MAP1LC3B & LAMP2 antibody & observed by immunofluorescent microscopy. (E,F) Representative bar graphs showing cART-mediated decreased LAMP2 puncta & decreased colocalization of MAP1LC3B & LAMP2. BAF—autophagosome fusion inhibitor, & rapamycin (RAP—autophagy inducer) were used as controls for autophagy flux. Data is from three independent experiments & is expressed as means ± SEM & were analyzed using one-way ANOVA. *, p < 0.05 vs. control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31569373), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - The cytoprotection of in vitro sIPC is diminished by autophagy inhibitors in RPTC cells. RPTC cells were subjected to: (1) control; (2) CCCP-R; (3) sIPC + CCCP-R in the absence or presence of chloroquine (20 μM) & 3-methyladenine (10 mM). Both inhibitors were used for 1-h pretreatment & during 2-h recovery from prolonged CCCP treatment. Cells were collected for morphological & immunoblot analyses. (a) Representative images of phase contrast & fluorescence microscopy showing cellular & nuclear morphology of apoptosis. Scale bar: 200 μm. (b) Quantification of cell apoptosis. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the control group; #, P < 0.05, significantly different from CCCP-R group. (c) Analysis of apoptosis inhibitory efficiency by sIPC. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the group without inhibitors. (d & e) Immunoblots of LC3B & cleaved CASP3. ACTB was used as a loading control. The molecular mass marker lanes were labelled as kDa. For densitometric analysis of cleaved CASP3, after normalization with ACTB, the protein signals of the control were arbitrarily set as 1, & the signals of other conditions were normalized to the control to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the control group; #, P < 0.05, significantly different from CCCP-R group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31066324), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - TFEB overexpression in macrophages induces the autophagy markers LC3 & p62 & restores their co-localization in atherosclerotic aortic roots.(a,b) Representative immunofluorescence images of atherosclerotic aortic roots (2 months' western diet) from control & mφTFEB-TG mice (ApoE-null background) stained with antibodies against TFEB (a), TFEB & MOMA-2 (b; scale bar, 50 μm). (c) Quantification of the average TFEB intensity & co-localization with nuclear marker DAPI (n=4-5 mice per group). (d) Representative immunofluorescence images of atherosclerotic aortic roots from control & mφTFEB-TG mice stained with p62 & LC3 (scale bar, 50 μm). (e) Quantification of the p62 & LC3 average intensity from control & mφTFEB-TG-stained roots (n=13–14 mice per group). (f) Representative pseudocolour image of these p62/LC3 images (green represents co-localization) & graph depicting the increased p62/LC3 correlation seen in a representative mφTFEB-TG as compared to a control lesion (scale bar, 50 μm). (g) Quantification of the p62/LC3 co-localization from control & mφTFEB-TG-stained roots shown (n=13–14 mice per group). (h,i) FACS analysis of aortic macrophages isolated from atherosclerotic aortas of Control or mφTFEB-TG mice (western diet-fed ApoE-KO background, n=3–4 pooled aortas) & stained for either (h) p62 & LC3, or (i) Lamp2 & LC3 antibodies (per cent of macrophages expressing each marker is shown below plots). For all graphs, data are presented as mean±s.e.m. *P<0.05, ***P<0.001, two-tailed unpaired t-test. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms15750), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - Keap1 co-localizes with SQSTM1-immunopositive sarcoplasmic aggregates in AVM muscle. a-c. Only focal & minimal co-localization of LC3 (a) & Keap1 immunofluorescence (b) is seen in a representative toxic AVM sample (colchicine-treated subject #31); merged panel is shown in (c) (LC3, red; Keap1, green). d-f. In the same specimen, there is extensive co-localization of SQSTM1 (d) & Keap1 immunofluorescence (e); merged panel is shown in (f) (SQSTM1, red; Keap1, green). g-i. Similar to toxic AVM specimens, essentially no co-localization of LC3 (g) & Keap1 immunofluorescence (h) is seen in a representative IBM muscle biopsy (subject #46); merged panel is shown in (i) (LC3, red; Keap1, green). j-l. In the same specimen, there is extensive co-localization of SQSTM1 (j) & Keap1 immunofluorescence (k); merged panel is shown in (l) (SQSTM1, red; Keap1, green). Scale bar, 25 μm Image collected & cropped by CiteAb from the following publication (//actaneurocomms.biomedcentral.com/articles/10.1186/s40478-016-0384-6), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Glucose deprivation (GD) induces neuronal cell death.Time course of cell viability & cell death after GD was estimated by MTT & LDH assays, respectively. Bars depict rates of MTT reduction (A) & LDH release (B) time dependent manner after GD. MTT data represent relative percentages compared with controls (*p<0.05, **p<0.001, n = 3). LDH release from neuro2a cells was calculated as fold increase from the LDH release in controls (*p<0.01, **p<0.001, n = 3). (C) Levels of LC3 & p62 expression, cleaved-caspase 3 & PARP cleavage were determined at indicated times after GD by immunoblot analysis. Tubulin was used as loading control. (D) Quantitative analyses of the immunoblots are shown in (C). The amount of each protein was normalized against the amount of tubulin. Data represent the mean ± SD for each condition (*p<0.05, **p<0.001, n = 3). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0076466), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Measurement of dysregulated autophagy by fluorescent microscopyOVCAR3 cells were treated with the indicated drugs for 24 hours & then fixed & immunostained for p62 (red) & LC3 (green), with a DAPI costain (blue). Confocal z-stacks were flattened for image analysis to capture all punctate area. A. Doses were: chloroquine (10 μM), nelfinavir (10 μM), rapamycin (10 nM), dasatinib (50 nM), & all were combined for Necramed. B. Similar immunostaining as in (A) Punctae were quantified for size & number per cell by ImageJ with at least 40 cells per condition. LC3 channel was analyzed for autophagosomes, & the p62 channel for sequestosomes. Doses were same as in (A), but with chloroquine & nelfinavir reduced to 5 μM. *p < 0.05, ***p < 0.001 by t-test. All error bars are s.e.m. C. OVCAR3 cells with a virally integrated mCherry-GFP-LC3 construct were studied by live microscopy, for the number of hours indicated following Necramed (doses as in (A)) or control treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26418751), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Rop-DPRL combined with CR inhibits tumor growth. (A) Fluorescent imaging of major organs & tumor. Tumor-bearing mice were injected with cy5-labeled liposome or unlabelled liposome. The tumor & major organs from control or drug-treated mice were excised at 12 h or 24 h. (B) Fluorescent image of tumor sections. Tumor from control or Rop-DPRL treated mice were excised at 12 h. Tumor frozen sections were observed under confocal microscope. Scale bar = 20 µm. (C) Immunofluorescence staining of tumor sections for the autophagosome marker LC3, P62 & LAMP1. (D) Image of representative tumors. (E) Change in tumor volume. (F) Tumor weight. (G) TUNEL staining of tumor sections. Nuclei were stained with Hoechst. Scale bar = 20µm. (H) Body weight changes of mice under different treatments. CR: Calorie restriction. Arrows: Enlarged lysosomes. Data are presented as the mean ± SEM, ** p < 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32308756), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - SsD & SsA inhibit autophagy in HeLa cells. a Structures of SsA, SsC, & SsD. b SsD (15 μM) & SsA (30 μM), but not SsC, significantly increased LC3-II yellow puncta formation but did not significantly affect LC3-II red-only puncta in RFP-GFP-LC3-expressing HeLa cells. Scale bar = 10 μm. Quantification of LC3 yellow puncta/red puncta (%) is presented as the mean ± S.E., n = ~80 cells from 3 independent experiments. c Treatment of HeLa cells with SsD for 6 h induced the accumulation of both LC3-II & SQSTM1 in a dose-dependent manner. d SsD (15 μM) induced the accumulation of both LC3-II & SQSTM1 in HeLa cells in a time-dependent manner. e SsA (30 μM) induced the accumulation of both LC3-II & SQSTM1 in HeLa cells in a time-dependent manner. f Treatment with different concentrations of SsC for 6 h failed to induce the accumulation of either LC3-II or SQSTM1 in HeLa cells. g SsD (15 μM) reversibly inhibited autophagy Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30820356), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - PERK pathway-independent induction of prosurvival autophagy in KG1 LSC-like cells upon G9a inhibition. a, LC3-I/II & p62 expression, analyzed by western blotting, after cell treatment with BIX-01294 (15 μmol/L) in the absence or presence of bafilomycin A1 (BafA1) for 24 h. b, Confocal microscopy images of GFP-LC3-transfected cells after treatment with BIX-01294 (5 μmol/L). c, Apoptotic fractions, measured using flow cytometry, after cell treatment with BIX-01294 (15 μmol/L) in the absence or presence of the autophagy inhibitor 3-MA (5 mmol/L) or BafA1 (2 nmol/L) for 48 h. d, Protein expression, analyzed by western blotting with the indicated antibodies, after treatment of PERK siRNA-transfected & non-transfected cells with BIX-01294 (15 μmol/L) for 48 h. e, Protein expression, analyzed by western blotting with the indicated antibodies, after cell treatment with BIX-01294 (10 μmol/L) in the absence or presence of GSK2606414 (20 μmol/L) for 48 h. f, Protein expression, analyzed by western blotting with the indicated antibodies, after treatment of NRF2 siRNA-transfected & non-transfected cells with BIX-01294 (10 μmol/L) for 48 h. g, Levels of apoptosis, evaluated by flow cytometry, after cell treatment for 48 h with BIX-01294 (10 μmol/L) in the absence or presence of the PERK inhibitor GSK2606414 (10 μmol/L) or the autophagy inhibitor 3-MA (5 mmol/L), or BafA1 (2 nmol/L). h, Levels of apoptosis, evaluated by flow cytometry, after treatment of PERK siRNA- or control siRNA-transfected cells with BIX-01294 (10 μmol/L) in the absence or presence of 3-MA (5 mmol/L) or BafA1 (2 nmol/L) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32293500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Metabolic profiling & biochemical assay. (A) Relative abundance of the substrates in the glycolytic pathway & TCA cycle in ORP compared to OCL by targeted metabolic profiling. When compared with OCL heart, ORP hearts have significantly lower glucose-6-phosphate & fructose-6-phosphate (both are glycolytic metabolites), & significantly higher a-ketoglutarate, fumarate, malate, & citrate (all are TCA cycle metabolites). *P < 0.05 compared with OCL. See Table S6 for numerical data. (B) A schematic diagram summarizing the changes in metabolism by rapamycin in old heart. (C) Western blots of autophagic markers show no significant change of LC3 II/I, p62, or beclin-1 in cardiac aging. However, OCR has significantly lower p62 than that in OCL. #P < 0.05 compared with OCL. (D) Both CR & RP significantly reduce the age-dependent increase in protein carbonyls (nmol mL−1). #P < 0.05 compared with OCL. (E). Both CR & RP significantly reduce the age-dependent increase in protein ubiquitination.*P < 0.05 compared with YCL & #P < 0.05 compared with OCL. n = 3–8. G6P: glucose 6-phosphate; G1P: glucose 1-phosphate; F6P: fructose 6-phosphate; F1P: fructose 1-phosphate; F16BP: fructose 1,6-bisphosphate; F26BP: fructose 2,6-biphosphate; G3P: glyceraldehyde 3-phosphate; DHAP:dihydroxyacetone phosphate; 2(3)-PGA: 2- or 3-phosphoglycerate; & PEP: phosphoenolpyruvate. Isomers of same molecular weight, that is, G6P versus G1P, F6P versus F1P, & F16BP versus F26BP, were not distinguishable by the LC-MS/MS-based metabolic profiling method. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/24612461), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - iNOS level is regulated by autophagy during LPS-stimulated condition. (A) Raw 264.7 cells were treated with LPS (200 ng/mL) for 12 h first, followed by removing the LPS & added CQ (30 µM), SAR (1 µM), Torin (1 µM), Rap (1 µM) for another 12 h. CTRL is blank control group with DMSO treatment. (B) Western blot analysis of iNOS in Raw 264.7 cells under LPS treatment. (C) Western blot analysis of p62 in Raw 264.7 cells under LPS treatment. (D) Raw 264.7 cells were treated LPS (200 ng/mL) for 12 h, then removed LPS added CHX (1 µg/mL) & autophagy related drugs for another 12 h. (E) Level of iNOS in Raw 264.7 cell after CHX treatment. (B–E) Values were analyzed from three individual experiments by GraphPad Prism, each experiment conducted three times. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars (mean ± SEM). One-way ANOVA with Turkeys as post hoc tests. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31618870), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Autophagic pathway regulates inflammasome activation in blood-derived monocytes stimulated with M. leprae. (A–C) Monocytes were purified from healthy donors’ peripheral blood mononuclear cells by 2-h adherence & stimulated with 10 µg/mL M. leprae (ML) in the presence or absence of autophagy regulators for 18 h. Autophagy was triggered with 200 ng/mL rapamycin (RP), & inhibited with 100 µM chloroquine (CQ, autophagic flux blocker) & 10 µM 3-methyladenine (3-MA). (A) Monocytes stimulated with M. leprae, RP, CQ, & 3-MA were fixed & stained with anti-NLRP3 (red), anti-LC3B (green) antibodies & DAPI (blue). Monocytes stimulated with M. leprae increase the number of LC3-II-decorated autophagosomes (puncta) per cell, which is reforced by RP addition, & reverted by 3-MA. Autophagy blocking with CQ & 3-MA was able to increase NLRP3 specks numbers per cell in relation to the well stimulated with M. leprae & RP. Immunofluorescence images were quantified & bars represent the mean values of the number of LC3 puncta & NLRP3 specks per cell ± SEM (n = 3). *(in relation to NS) or #(indicated by the dashes) p < 0.05. Scale bar: 10 µm. (B) Purified mRNAs from monocyte cultures stimulated with M. leprae, the autophagy blocker 3-MA, & RP, as a control of autophagy activation, were analyzed by RT-qPCR for LC3B & IL1B. Bars represent the mean of the fold change values in relation to the non-stimulated controls of three independent experiments with similar results. (C) The IL-1 beta , IL-6, & TNF levels in the monocyte cultures supernatants were assessed by ELISA. Bars represent the fold change values in relation to the non-stimulated controls ± SEM (n = 3). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29915584), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Melatonin induced LC3 aggregation & conversion. a GFP-LC3 aggregation & conversion in U87MG cells stably expressing an exogenous fusion protein, green fluorescent protein (GFP) & microtubule-associated light chain 3 (LC3) (U87-GFP-LC3), after treatment with DMSO (control), rapamycin (RAP 200 nM), melatonin (MEL 1 mM) or agomelatine (AGO 2 μM) for 24 h (×40); b U87MG cells & c A172 cells treated with DMSO (control), rapamycin (RAP 200 nM), melatonin (MEL 1 mM), agomelatine (AGO 2 μM) & luzindole (LUZ 5 μM/L) for 24 h were fixed & analyzed for endogenous LC3 dots by immunofluorescence using anti-LC3 antibodies (green), DAPI (blue) staining shows the nuclei (×40). d Stable U87-GFP-LC3 cell lines & GFP in vector control. U87MG cells stably expressing an exogenous fusion protein, green fluorescent protein (GFP) & microtubule-associated light chain 3 (LC3) (U87-GFP-LC3) (×40) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31870319), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Autophagy induction by atorvastatin in T24 human bladder cancer cells. (A) Immunocytochemistry for the co-localization of LC3 puncta (green) & Lysotracker (red, demarcation for the lysosome) after atorvastatin treatments (30 μM) for 24 h in T24 cells. Rapamycin treated T24 cells were the positive control of autophagy induction. 4′,6-diamidino-2-phenylindole (DAPI) was used for nucleus staining. Scale bar = 100 μm; (B) High magnification view of immunocytochemistry for the co-localization LC3 puncta (green) & Lysotracker after atorvastatin treatments (30 μM) for 24 h in T24 cells at the single-cell level. Scale bar = 50 μm; (C) Western blot analysis of autophagosome formation markers p62/SQSTM1, LC3-I & LC3-II in untreated (control) & atorvastatin (30 μM) treated T24 cells. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/15/5/8106), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Increased LC3B-positive puncta correlated positively with vascular remodeling during AngII-induced hypertension. (A) Western blot analysis of LC3B-II expression in aortas from sham or AngII-infused mice at different time points after operation. **P < 0.01 vs. the corresponding sham group, Student's t-test. n = 10 mice/group. (B) Representative immunofluorescence staining of LC3B (green) & alpha -SMA (red) in thoracic aorta section. Nuclei were stained with DAPI. Scale bars, 20 µm. (C) Quantification of LC3B puncta. Five random fields (2,500 µm2/filed) were measured in one section. Statistical significance was determined by one-way ANOVA. **P < 0.01 vs. sham, n = 12 sections from six mice per group. (D-G) Structural parameters of mouse aorta from sham & hypertensive mice. WD, wall diameter; LD, lumen diameter; CSA, cross-sectional area. *P < 0.05, **P < 0.01 vs. the corresponding sham group, Student's t-test. n = 10 mice/group. (H) Correlation between changes in LC3B puncta & CSA values. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32226533), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB100-2220] - Representative images of the variation of LC3 staining associated with cavities in SDH activity & mitochondrial COX-1 staining in PAD gastrocnemius muscle sections. First column shows succinate dehydrogenase (SDH) activity (dark fibers are type I, intermediate fibers are type IIa, & light fibers are type IIa/x), second column shows mitochondrial COX-1 protein (green), third column shows LC3 (red) & fourth column shows COX-1 & LC3 merged. Examples of normal fibers with no LC3 staining or COX-1 cavities (rowA), punctate LC3 staining where a COX-1 cavitiy is forming (row B), elevated LC3 staining with low COX-1 staining (rowC), LC3 accumulation in areas lacking COX-1 staining (row D), & LC3 plaque in the center of an SDH/COX1 cavity (COX-1; rowE). Arrows within the same row point to areas of LC3 accumulation in same fiber. Scale bar = 100 µM Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27687713), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Mechanistic analysis of autophagic inhibition in ATO-treated HepG2. a The viability of HepG2 & NB4 cells after incubation with ATO at various concentrations for 48 h (n = 3; error bars are s.d.). Upper: Confocal images of NB4 or HepG2 cells after being stained with calcein AM. Scale bars: 10 μm. b The viability of HepG2 & NB4 cells after incubation with CQ, ATO, or CQ–ATO mixture for 48 h (n = 3). ##P < 0.01 by t-test, significantly different from ATO. ns not significant. c Immunoblots of autophagy-related proteins LC3-II, p62 (left); semi-quantified analysis (n = 3) in NDs, ATO, or NDs–ATO mixture-treated cells (right). GAPDH was used as the loading control. Normalized band densities were shown below each band. d Cell viability & Caspase-3 activity after incubation with ATO or NDs–ATO mixture for 48 h (n = 3). ##P < 0.01 by t-test, significantly different from ATO. e Immunostaining of LC3 in NDs, ATO, or NDs–ATO mixture-treated cells (left). Scale bar: 20 μm. TEM images of ATO or NDs–ATO mixture-treated cells (right). Scale bars: 1 μm. Typical structures of autophagosomes induced by ATO & autolysosome containing NDs induced by NDs–ATO mixture are indicated with green or blue arrows, respectively (magnification). Vacuoles inside cells are indicated with red arrows (magnification). Scale bars: 500 nm. Error bars are s.d. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30341298), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - Rop-DPRL impairs autophagy. (A) Fluorescent images of GFP-LC3-HeLa cells treated with 1.5 mM ropivacaine for 0-24 h, or starvation for 0-24 h, & stained with 75 nM LysoTracker Red. Scale bar = 5 µm. (B) Western blot & (C) statistical results of P62, immature & mature cathepsin D (CathD), LC3I, LC3II, & beta -actin. HeLa cells were treated with ropivacaine or starvation for 0-24 h. (D) Western blot & (E) statistical results of P62, LC3I, LC3II, & beta -actin. HeLa cells were treated with PBS, CQ (50 µM), ropivacaine, or CQ+Rop for 24 h. (F) Fluorescent images of GFP-LC3-HeLa cells treated with empty liposome or Rop-DPRL for 24 h & stained with 75 nM LysoTracker Red. Scale bar = 5 μm. (G,I, K & M) Western blot & (H,J,L & N) statistical results for P62, LC3I, LC3II & beta -actin. Cells were treated with empty liposome, CQ, Rop-DPRL, starvation or indicated combination for 24 h. Data are presented as the mean ± SEM, *p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significant difference, Rop: ropivacaine, starv: starvation. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32308756), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: LC3B Antibody - BSA Free [NB100-2220] - PERK pathway-independent induction of prosurvival autophagy in KG1 LSC-like cells upon G9a inhibition. a, LC3-I/II & p62 expression, analyzed by western blotting, after cell treatment with BIX-01294 (15 μmol/L) in the absence or presence of bafilomycin A1 (BafA1) for 24 h. b, Confocal microscopy images of GFP-LC3-transfected cells after treatment with BIX-01294 (5 μmol/L). c, Apoptotic fractions, measured using flow cytometry, after cell treatment with BIX-01294 (15 μmol/L) in the absence or presence of the autophagy inhibitor 3-MA (5 mmol/L) or BafA1 (2 nmol/L) for 48 h. d, Protein expression, analyzed by western blotting with the indicated antibodies, after treatment of PERK siRNA-transfected & non-transfected cells with BIX-01294 (15 μmol/L) for 48 h. e, Protein expression, analyzed by western blotting with the indicated antibodies, after cell treatment with BIX-01294 (10 μmol/L) in the absence or presence of GSK2606414 (20 μmol/L) for 48 h. f, Protein expression, analyzed by western blotting with the indicated antibodies, after treatment of NRF2 siRNA-transfected & non-transfected cells with BIX-01294 (10 μmol/L) for 48 h. g, Levels of apoptosis, evaluated by flow cytometry, after cell treatment for 48 h with BIX-01294 (10 μmol/L) in the absence or presence of the PERK inhibitor GSK2606414 (10 μmol/L) or the autophagy inhibitor 3-MA (5 mmol/L), or BafA1 (2 nmol/L). h, Levels of apoptosis, evaluated by flow cytometry, after treatment of PERK siRNA- or control siRNA-transfected cells with BIX-01294 (10 μmol/L) in the absence or presence of 3-MA (5 mmol/L) or BafA1 (2 nmol/L) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32293500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - TLR4 mediates Hsp70/Hsp90-induced catabolic response in myotubes. a Hsp70 & Hsp90-induced catabolic response in myotubes is dependent on TLR4. C2C12 myoblasts were transfected with siRNA as indicated. After differentiation, myotubes were treated with rHsp70 & rHsp90 & analyzed for catabolic response as described above. b LLC-released EVs induce muscle catabolism in C2C12 myotubes through TLR4. C2C12 myotubes with either TLR2 or TLR4 knockdown were treated with EVs isolated from LCM (AchE 6 mU) & analyzed for catabolic response. c CD9-positive EVs activate TLR4 in reporter cells. EVs isolated from LCM were subjected to immunoprecipitation (IP) using an antibody against CD9 with pre-immune IgG as control. Resulting pellet & supernatant were used to treat TLR4 reporter cell line HEK-Blue hTLR4 for 24 h, & compared with original EVs. TLR4 activation was measured as SEAP activity. Data (n = 3) were analyzed by analysis of variance. * denotes a difference (P < 0.05) Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-017-00726-x), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB100-2220] - cIAP2 & XIAP regulate autophagosome fusion, but not cIAP1.a MEFs expressing mCherry-GFP-LC3b were transfected with siRNA against either cIAP1, cIAP2 or XIAP. Cells were analysed with the microscope & the number of mCherry+, & GFP + puncta/cell were calculated & the ratio of GFP + /mCherry + puncta is indicated. Shown are the means & the error bars represent the SEM of at least three independent experiments. Westerns show efficient knockdown of cIAP1 & XIAP expression. cIAP2 siRNA efficiency was determined by real time PCR as shown in the graph below the westerns. b Wild type & cIAP2−/− XIAP−/− dermal fibroblasts expressing mCherry-GFP-LC3b were treated analysed on the microscope & mCherry+, & GFP + puncta/cell were calculated. The ratio of GFP+/mCherry + puncta is indicated. c Wild type & cIAP2−/− XIAP−/− dermal fibroblasts were left in complete media (CM) or starved for 2 h in EBSS. Cells were lysed & proteins analysed by western blot for XIAP, LC3, & Actin. cIAP2−/− was confirmed by PCR due to lack of effective antibodies for mouse cIAP2 (see supplemental Fig. 2). d Immunofluoresence showing accumulation of LC3 in starved cIAP2−/− XIAP−/− dermal fibroblasts. Wild type & cIAP2−/− XIAP−/− dermal fibroblasts were incubated in complete media or starved in EBSS for 2 h, then fixed & stained for LC3 (green channel) & LAMP2 (red channel). Nuclei are stained blue with Hoechst. Shown in upper panels are overviews. Lower panels show zoomed in regions indicated in the upper panels Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29743550), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Enhanced mTORC2 signaling is sufficient to protect MSC from calcification. Flag-tagged rictor or green fluorescent protein (GFP) as a control were introduced into MSC by lentiviral transfer. Cells were cultured for 21 days under control conditions, with osteoblast induction medium (OM) & vehicle, or OM & 20 nM Rapa as indicated. (A) Western blot analyses of rictor & flag confirming overexpression of rictor due to gene transfer, downstream targets of the two mTOR complexes (mTORC1: pp70-S6KSer389; mTORC2: pAKTSer473), inhibition of autophagy (LC3B II, p62), cellular senescence (p16INK4a), & apoptosis-related proteins (Bcl-2: negative regulator of apoptosis; cleaved caspase 3 (cl casp 3): executioner caspase). Representative western blots from six independent experiments are shown. For densitometric quantification, band intensities were normalized to GAPDH as a loading control. Control (no OM) was set to 1. (B) Lactate dehydrogenase (LDH) activity in cell culture supernatants normalized to total protein concentrations was determined to quantify cell death. Control was set to 1. (C) Alkaline phosphatase (ALP) activity normalized to total protein concentrations was measured after 7 days of incubation. (D) Calcium deposition was quantified with the ortho-cresolphthalein method & normalized to total protein concentrations. Bars represent mean + SEM, *P < 0.05, **P < 0.01, ***P < 0.001. Numbers of independent experiments are indicated at the bottom of each graph. (E) Alizarin red S staining of calcium deposits. Representative experiment out of six, phase contrast microscopy, original magnification x200, scale bar = 100 µm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31882658), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB100-2220] - OSI induces autophagy by activating LKB1/AMPK signaling in CRC cells.a Immunoblotting analysis of AMPK, phosphorylated AMPK (Thr172) & LC3 levels in CRC cells treated with indicated concentration of OSI for 24 h. b Immunohistochemistry analysis of AMPK phosphorylation levels in xenograft tissues. Scale bar, 50 μm. c Relative intensity of phosphorylated AMPK staining in (b). d CRC cells were transfected with siScramble or siAMPK for 24 h & followed by treatment with or without 5 μM for another 24 h. LC3 & phosphorylated AMPK (Thr172) levels were detected by immunoblotting. e CRC cells were transfected with empty vector or DN-AMPK plasmid for 24 h, followed by treatment with or without 5 μM OSI for another 24 h. LC3 & AMPK phosphorylation levels were determined by immunoblotting. f CRC cells were treated as in (d), the endogenous LC3 puncta in CRC cells were assessed by immunofluorescence. Scale bar, 10 μm. g The number of LC3 puncta per cell in (f). h CRC cells were treated as in (e), the endogenous LC3 puncta in CRC cells were assessed by immunofluorescence, Scale bar, 10 μm. i The number of LC3 puncta per cell in (h). j Immunoblotting analysis of phosphorylated LKB1 & phosphorylated CaMKK beta levels in CRC cells treated with or without 5 μM OSI. Data are presented as mean SEM, Student’s t-test, & are representative of three independent experiments. ***P < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31409796), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: LC3B Antibody - BSA Free [NB100-2220] - Cathepsin inhibitors promote neuronal survival after glucose reperfusion (GR).After 3 h GD, neuro2a cells were replaced by 20; pepstatin A (PA, 10 uM), leupeptin (Leu, 10 uM) or both (PA+Leu). (A) Cell viability was assessed using the MTT assay. MTT mitochondrial reduction was shown as a relative percentage of the MTT values at the indicated time point (3 h GD/48 h GR) from three independent experiments ± SD (*p<0.05, **p<0.001). (B) Changes of protein level by cathepsin inhibitors were analyzed by immunoblot analysis. Proteins from cell lysates were fractionated on SDS-PAGE; blots were probed with anti-LC3, anti-p62, anti-cleaved caspase-3, & anti-PARP antibodies. Tubulin was used as loading control. (C) The graphs represent relative band density of each protein shown in (B). The amount of each protein was normalized against the amount of tubulin. Data represent the mean ± SD (*p<0.05, n = 3). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0076466), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Reactivity:

Hu, Mu, Rt, Po, Al, Av, Ba, Bv, Ca, Ch, ChHa, Gp, Ha, In, Pm, Pm, Rb, SyHa, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Al, Av, Ba, Bv, Ca, Ch, ChHa, Gp, Ha, In, Pm, Pm, Rb, SyHa, Ze

Applications:

, , , , , , , , , , , ,

Applications: , , , , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-2220AF350
Alexa Fluor 405	NB100-2220AF405
Alexa Fluor 488	NB100-2220AF488
Alexa Fluor 532	NB100-2220AF532
Alexa Fluor 594	NB100-2220AF594
Alexa Fluor 647	NB100-2220AF647
Alexa Fluor 700	NB100-2220AF700
Alexa Fluor 750	NB100-2220AF750
Alexa Fluor Plus 405	NB100-2220AFP405
Alexa Fluor Plus 488	NB100-2220AFP488
Alexa Fluor Plus 555	NB100-2220AFP555
Alexa Fluor Plus 594	NB100-2220AFP594
Alexa Fluor Plus 647	NB100-2220AFP647
Alexa Fluor Plus 680	NB100-2220AFP680
Allophycocyanin	NB100-2220APC
Biotin	NB100-2220B
CoraFluor 1	NB100-2220CL1
DyLight 350	NB100-2220UV
DyLight 405	NB100-2220V
DyLight 488	NB100-2220G
DyLight 550	NB100-2220R
DyLight 594	NB100-2220DL594
DyLight 650	NB100-2220C
DyLight 680	NB100-2220FR
DyLight 755	NB100-2220IR
FITC	NB100-2220F
HRP	NB100-2220H
Janelia Fluor 525	NB100-2220JF525
Janelia Fluor 549	NB100-2220JF549
Janelia Fluor 646	NB100-2220JF646
Janelia Fluor 669	NB100-2220JF669
PE	NB100-2220PE
PerCP	NB100-2220PCP
mFluor Violet 450 SE	NB100-2220MFV450
mFluor Violet 500 SE	NB100-2220MFV500
mFluor Violet 610 SE	NB100-2220MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-2220SS

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NB100-105

HIF-1 alpha Antibody (H1alpha67)

(45 Reviews)

(1064 Publications)

0.1 ml

NB100-105

HIF-1 alpha induction by CoCl2 on Caki-1 cell lysate. WB image submitted by a verified customer review.

<b>Biological Strategies Validation and Genetic Strategies Validation.</b>HIF-1 alpha was detected in immersion fixed DFO treated Hela cells (left) but was not detected in HIF-1 knockout HeLa cells (right) using Mouse Anti-human HIF-1 alpha monoclonal antibody (Catalog # NB100-105) at 25 ug/mL for 3 hours at room temperature. Cells were stained using a NorthernLights (TM) 557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to nuclei.

<b>Biological Strategies Validation and Genetic Strategies Validation.</b>Naive CD4 T cells from WT, VHL-deficient (Vhl KO), or HIF-1 alpha-deficient (HIF-1 alpha KO) mice were differentiated under IL-22-skewing conditions for a total of 60 h. Some cells remained at normoxia for the duration of the culture (N); others were at normoxia for 35 h and then hypoxia (1% O2) for 24 h (H). At 60 h, nuclear extracts were harvested, and HIF-1 alpha and Lamin B1 levels were analyzed by Western blot. Image from verified customer review.

Staining of HIF-1 alpha in human kidney using NB100-105. Renal tubular epithelium showed moderate membranous, cytoplasmic and nuclear staining, and glomeruli showed faint to moderate nuclear staining.

Analysis using the Alexa Fluor (R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in multiple myeloma cells: H929 cells (0.5 x 10^6) were stained with Alexa Fluor 488(R) conjugated HIF-1 alpha antibody (NB100-105AF488). Image courtesy of Dr. Barbara Muz at Washington University in St. Louis School of Medicine.

HIF-1-alpha-Antibody-H1alpha67-Western-Blot-NB100-105-img0029.jpg

Analysis using the biotin conjugate of NB100-105. Staining of human glioblastoma multiforme.

<b>Biological Strategies Validation. </b>HIF-1 alpha was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line treated with DFOA using 1 ug/mL of mouse anti-HIF-1 alpha monoclonal antibody NB100-105. Cells were stained using a donkey anti-rabbit secondary antibody and counterstained with DAPI (blue).

<b>Biological Strategies Validation. </b>HIF-1-alpha-Antibody-H1alpha67-Western-Blot-NB100-105-img0032.jpg

Analysis using HIF-1 alpha Antibody on human throat cancer tissue. PFA fixed paraffin-embedded sections. Primary antibody dilution 1:100. Image from verified customer review.

<b>Biological Strategies Validation. </b>Analysis of HIF-1 alpha in human hepatocytes from cancer patient using anti-HIF-1 alpha antibody NB100-105. Western blot image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>Analysis using the HRP conjugate of NB100-105. Detection of 50 ug cobalt chloride induced COS-7 nuclear extracts (NB800-PC26) using NB100-105.

HIF-1-alpha-Antibody-H1alpha67-Western-Blot-NB100-105-img0030.jpg

HIF-1-alpha-Antibody-H1alpha67-Western-Blot-NB100-105-img0031.jpg

HIF-1-alpha-Antibody-H1alpha67-Immunohistochemistry-NB100-105-img0033.jpg

Analysis using the Alexa Fluor(R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in H929 cells using HIF-1 alpha antibody. Flow cytometry image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>Effects of one- and five-dosage dry needling (1D, 5D) on iNOS, HIF-1 alpha , COX-2, and VEGF expressions in a needling-treated muscle. (a) Representative Western blot images. The quantification of the protein levels for (b) iNOS, (c) HIF-1 alpha , (d) COX-2, and (e) VEGF. Values are expressed as mean ± SD. *Indicates the significant difference (P < 0.05) between the sham groups (s1D and s5D). #Represents the significant difference (P < 0.05) between the 1D and 5D groups. DN: dry needling.

<b>Biological Strategies Validation. </b>Hypoxia is implicated in the adaptive response after short-term bevacizumab treatment. Expression of HIF-1 alpha in pulmonary tumor nodules of the four groups. (A) A representative western blot is shown. beta -actin was used as a loading control. (B) While most tumors showed little expression of HIF-1 alpha protein in PBS and cisplatin groups, mice that received bevacizumab and bevacizumab + cisplatin therapy showed a markedly increased level of HIF-1 alpha expression.. *P < 0.05, **P < 0.01.

<b>Biological Strategies Validation. </b>Effects of F2 on tumor-related biomarkers in vivoImmunohistochemical analysis of tumor-related proteins (HIF-1 alpha , VEGF, MMP-2, CD31 and KI67) in tumor sections from U87 xenograft mice treated with F2 (mg/kg i.g.). Red arrows indicate the related protein detected in the tissue samples. Values are means ± SEM of three experiments. * P<0.05 compared with DDW group.

<b>Biological Strategies Validation. </b>AAM inhibits hypoxia-induced VM formation involving HIF-1 alpha and MMP2. The HCT-116 (A) and LoVo (B) cells were treated with different concentrations of AAM under hypoxia and normoxia; the protein expression of HIF-1 alpha and MMP2 was detected by western blotting. (C, D) The mRNA expression of HIF-1 alpha and MMP2 was measured by RT-PCR (*P < 0.05, **P < 0.01, ***P < 0.001).

Immunohistochemical analysis of STC1 expression in esophageal cancer. (A) Normal squamous epithelium was negative for STC1 expression. (B) Normal gastric glands showed positive staining for STC1, which was used as a positive control. (C) Positive staining for STC1 in esophageal squamous cell carcinoma (ESCC). (D) Negative staining for STC1 in ESCC. (E) Positive staining for HIF-1 alpha in ESCC. (F) Positive staining for p53 in ESCC. Magnification, ×100.

Simple Western: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of hypoxia on human airway epithelial cells (A549).A549 cells were subjected to hypoxia for 6, 24, and 48 hours. Cell lysates were collected and analyzed for the expression levels of various hypoxic markers by qPCR (n = 4). (A-C) The expression levels of HIF-1 alpha (A), GLUT-1 (B), and VEGFA (C) were measured. (D-G) Additionally, whole-lung lysate was used for Western blots to determine the protein expression of HIF-1 alpha, VEGFA, and FIH-1. After analyzing the data, we used the unpaired t test with Welch's correction to determine its statistical significance. *P < 0.05, ***P < 0.001, ****P < 0.0001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/37737265 ), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

The HIF-1 alpha -BNIP3 pathway was activated during hypoxia. (a) Analysis of mitophagy-related protein expression in Neuro-2a cells by immunoblotting. Neuro-2a cells were exposed to hypoxia and/or high glucose for 48 h. Total cellular extracts were analysed by immunoblotting with antibodies against HIF-1 alpha , BNIP3, PINK1 & TOMM20. (b) The ImageJ densitometric analysis is shown. “C” represented control conditions (25 mM glucose), “G” represented high glucose conditions (75 mM glucose). The results are expressed as the mean ± SEM from three independent experiments. Two-way ANOVA, **p < 0.01, *p < 0.05, Hypoxia vs Normoxia. Full-length blots are presented in Supplementary Fig. S2. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-20162-1), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Inhibition of tumor growth in vivo by various treatments. (a) Macroscopic view of mouse tumors at the end of the study. (b) Tumor growth curves from day 0 to 40, with tumor sizes measured every 5 days, in various treatment groups as indicated. (c) Wet weight of tumors measured at the end of the study. (d) Protein expression of HIF-1 alpha examined by western blot in PC-3 xenografts exposed to various treatments. Data were presented as mean ± SD (n = 6). *p < 0.05 versus control group; #p < 0.05 versus si-HIF-1 alpha or DDP group. The original blots are presented in Supplementary Figure 6. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-07973-4), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - AAM inhibits hypoxia-induced VM formation involving HIF-1 alpha & MMP2. The HCT-116 (A) & LoVo (B) cells were treated with different concentrations of AAM under hypoxia & normoxia; the protein expression of HIF-1 alpha & MMP2 was detected by western blotting. (C, D) The mRNA expression of HIF-1 alpha & MMP2 was measured by RT-PCR (*P < 0.05, **P < 0.01, ***P < 0.001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32499699), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Muscle repair following eccentric contraction–induced injury is concomitant with dynamic alterations of HIF2A & HIF1A expression in SCs.(A–C) Representative images of EDL myofibers from injured muscles at various time points (n >50 myofibers from 3 mice/group/time point) & stained for Pax7, DAPI, & EdU (A), HIF2A (B), or HIF1A (C). Scale bars: 20 μm. Arrowheads indicate SCs. (D) Number of Pax7+ SCs per myofiber at various time points. (E) Percentage of EdU+ SCs at various time points. (F) Percentage of HIF2A+ SCs at various time points. (G) Percentage of HIF1A+ SCs at various time points. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - mTORC2 not mTORC1 regulates HIF‐2 alpha , GOT1 in PDAC under prolonged hypoxia. (A & B) HIF‐2 alpha mRNA & protein levels determined by qRT‐PCR (mRNA) & Western blot (protein) after treatment of Panc‐1 & Capan‐2 cells with mTORC1 inhibitor rapamycin for 48 hrs at 3% or 1% O2. beta ‐Actin was used as loading control. (C & D) Panc‐1 & Capan‐2 cells were treated with mTORC1/mTORC2 inhibitor PP242 & cultured for 48 hrs at 20%, 3% or 1% O2, & HIF‐2 alpha , GOT1 mRNA & protein levels were determined by qRT‐PCR (mRNA) & Western blot (protein). beta ‐Actin was used as loading control. Data are presented as mean ± S.D. from three independent experiments. *P < 0.05, **P < 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28544376), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Murine tumor cells express connexin 43 (Cx43) & hypoxic-induced factor-1 alpha (HIF-1 alpha ). The expression of Cx43 & HIF-1 alpha was measured by Western blot analysis. beta -actin expression served as loading controls & total protein. Inserted values indicated relative protein expression in comparison with beta -actin. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/16/1/439), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Representative microscopy images of staining for hypoxia markers in prostate tissues (MO, 400×). A) HIF-1 alpha - notice the granular cytoplasmic immunoreactivity of the malignant epithelial cells. In this case, more than 50% of the glands stained. B) LOX - strong & diffuse nuclear immunoreactivity of the epithelial cells. C) CAIX - note a focal apical cytoplasmic immunoreactivity in epithelial cells. D) VEGFR2 - moderate nuclear & weak cytoplasmic expression of the epithelial cells Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28143503), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of renal medullary transfection of PHD2 plasmids on the levels of PHD2 & HIF-1 alpha in the renal medulla. (A) PHD2 mRNA levels. (B) Representative ECL gel documents of Western blot analyses depicting the protein levels of HIF-1 alpha . (C) Summarized intensities of the HIF-1 alpha blots (normalized to LS). *P < 0.05 versus others (n = 7–8). LS = low salt, HS = high salt, Ctrl = control vectors, PHD2 = PHD2 expression vectors, CoCl2 = sample from cells treated with CoCl2 as positive control. Renal medullary tissues were obtained at the end of blood pressure recording after 3 week high salt challenge. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/22686466), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Immunohistochemical analysis of STC1 expression in esophageal cancer. (A) Normal squamous epithelium was negative for STC1 expression. (B) Normal gastric glands showed positive staining for STC1, which was used as a positive control. (C) Positive staining for STC1 in esophageal squamous cell carcinoma (ESCC). (D) Negative staining for STC1 in ESCC. (E) Positive staining for HIF-1 alpha in ESCC. (F) Positive staining for p53 in ESCC. Magnification, ×100. Image collected & cropped by CiteAb from the following publication (//www.spandidos-publications.com/10.3892/or.2011.1607), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1alpha & CAIX expression in PDAC & adjacent normal pancreatic tissue. Adjacent normal exocrine pancreas with weak nuclear & cytoplasmic HIF-1alpha staining (a). Adjacent normal pancreatic tissue with weak membranous CAIX staining in the ducts & absent staining in exocrine pancreatic tissue (b). PDAC with weak (intensity 1) nuclear & cytoplasmic HIF-1alpha staining (c). PDAC with strong (intensity 3) nuclear & moderate (intensity 2) cytoplasmic HIF-1alpha staining (d). PDAC with weak to moderate (intensity 1–2) membranous CAIX staining (e). PDAC with strong (intensity 3) membranous CAIX staining (f) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29973215), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha staining within injured muscle area (representative figure).The animal was ventilated with 100% O2. 450 minutes after trauma, the Musculus gastrocnemius of the traumatized right hind limb was harvested & section of the muscle specimen was analyzed for HIF-1 alpha expression within the injured area. No significant staining for HIF-1 alpha of the muscle cells. Scale bar: 150 µm. Inlet: Positive staining for HIF-1 alpha expression by myeloid cells (brown), invading to the traumatized muscle. Scale bar: 10 µm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0111151), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha is activated in VSMCs during Ang II-induced vascular remodeling.WT mice were infused with saline or 1000 ng/kg/min Ang II for 28 days. a Immunofluorescence analysis of representative cross-sections of mice aortas for HIF1 alpha (red) & alpha -SMA (green), nuclei was stained with DAPI. VSMCs were isolated form WT mice & treated with 1 μM Ang II for 24 h. bHif1a mRNA was measured by qPCR analysis. c HIF1 alpha protein was detected by western blot. **P < 0.01, ***P < 0.001, n = 3 per group, statistical significance was determined by the unpaired t-test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31320613), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Hypoxia is implicated in the adaptive response after short-term bevacizumab treatment. Expression of HIF-1 alpha in pulmonary tumor nodules of the four groups. (A) A representative western blot is shown. beta -actin was used as a loading control. (B) While most tumors showed little expression of HIF-1 alpha protein in PBS & cisplatin groups, mice that received bevacizumab & bevacizumab + cisplatin therapy showed a markedly increased level of HIF-1 alpha expression.. *P < 0.05, **P < 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/22357313), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - DC101, evofosfamide, & low dose doxorubicin multimodal treatment of KP sarcomasA. Tumor volume of KP sarcomas. Groups were treated by intraperitoneal injection of control IgG 20 mg/kg 3 times per week, DC101 20 mg/kg 3 times per week, evofosfamide (Evo) 50 mg/kg 5 times per week, and/or low dose doxorubicin (Dox) 1 mg/kg 3 times per week. Photos B. & graphs C. of PCNA immunohistochemistry for proliferation, TUNEL immunofluorescence for apoptosis, CD31 immunohistochemistry for microvessel density, CD31 & TUNEL immunofluorescence for endothelial cell (EC)-specific apoptosis, & nuclear HIF-1 alpha immunohistochemistry for HIF-1 alpha activity. Bars represent standard deviation. **p < 0.05 compared to all other groups. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.10212), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Overexpression of HIF alpha proteins increases IL-1 alpha transcription in A549 & HeLa cells but not in WI-38 fibroblasts.(A) Western blot using anti-HIF-1 alpha or anti-HIF-2 alpha antibodies of nuclear fractions obtained from HEK-T293 cells transfected with the annotated vectors. WI-38 (B), A549 (C), or HeLa (D) cells were transfected with either control vector or with HIF-1 alpha (P402A/P564A) & HIF-2 alpha (P405A/P531A), & 24 h later were analyzed for IL-1 alpha & VEGF RQ of mRNA levels by real-time PCR. Graphs represent mean ± SEM of four independent experiments for each cell type. P values of HIF alpha -transfected cells vs. control (Ct) transfected are annotated in the graphs. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23049530), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effects of F2 on tumor-related biomarkers in vivoImmunohistochemical analysis of tumor-related proteins (HIF-1 alpha , VEGF, MMP-2, CD31 & KI67) in tumor sections from U87 xenograft mice treated with F2 (mg/kg i.g.). Red arrows indicate the related protein detected in the tissue samples. Values are means ± SEM of three experiments. * P<0.05 compared with DDW group. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.19654), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Immunohistochemical labeling of normal brain & glioma tissueRepresentative images of normal temporal lobe, anaplastic astrocytoma grade II & grade III (AAII & AAIII), & three GBMs with low, medium, & high labeling of (A) MAOB, (B) HiF-1 alpha , & (C) GFAP are shown, along with (D) Sp1 & e) Sp3. The antibodies were detected with DAB & the nuclei were labeled with hematoxylin. Each image in a column was taken from a single slide, where each tissue slice was treated identically & imaged using the same microscope setting & with no electronic image manipulation. Arrows indicate nuclear HiF-1 alpha . Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.6582), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Expression of PACAP, VIP, VIPRs, HIF-1 alpha , HIF-2 alpha , & EGFR in glioblastoma multiforme (GBM). (A) Representative immunoblot of PACAP & VIP precursor peptides & PAC1R, VPAC1R, & VPAC2R expression on frozen glioblastoma sample. (B) Representative immunoblot & photomicrographs of signals detected by antibodies direct against HIF-1 alpha , HIF-2 alpha , & EGFR in a frozen glioblastoma sample. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/Article/10.3389/fphar.2016.00139/abstract), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Regulation of cartilage degradation by HIF-1 alpha . (A) Amount of glycosaminoglycans released into the medium determined by the dimethylmethylene blue assay during 3 days culture of Hif1afl/fl (Cntl) & Col2a1-CreERT2;Hif1afl/fl (KO) femoral heads treated with or without 10 ng/mL IL-1 beta under the hypoxic condition (3% O2). Tamoxifen induction was performed three days before sacrifice. Bars show the mean ± SD of three samples per group. *P < 0.05. (B) Immunofluorescence of Mmp13, Hif2a, & Hif1a in the Cntl & KO femoral head cartilage after 3-day culture with or without 10 ng/mL IL-1 beta . Scale bars, 50 µm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32214220), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha /PFKFB3 is upregulated in beta -cells of HIP rats & humans with type 2 diabetes. Representative immunofluorescence images of islets from a WT & HIP rats & b non-diabetic (ND) & T2D patients stained for PFKFB3 (red), insulin (green), & nuclei (blue). c Frequency of PFKFB3 positive beta -cells in HIP versus WT rats (left panel) & T2D versus ND patients (right panel). d Representative Western blot of PFKFB3 & HIF1 alpha levels in nuclear-enriched- & whole cell extracts from non-diabetic (ND) & T2D donor islets. Data are presented as mean ± SEM, n = 3 independent biological samples for each group. Statistical significance was analyzed by Student t-test (*p < 0.05, ***p < 0.001) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31213603), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha & HIF-2 alpha expression is differentially modulated by Hsp90 inhibition in CCRCC cells. UMRC2 & 786-O cells were treated for 20 h with the Hsp90 inhibitors 17-AAG (1 μM), EC154 (100 nM), the HDAC inhibitor LBH589 (100 nM), or DMSO vehicle control. Nuclear protein was analyzed for HIF-1 alpha & HIF-2 alpha & cytosolic protein was analyzed for RACK1 by SDS-PAGE & Western blot. Topoisomerase II & GAPDH were used as nuclear & cytosolic loading controls, respectively. Bands corresponding to HIF-1 alpha & HIF-2 alpha Western blots were quantified, normalized to TOPOII, & are expressed as a percentage of control samples. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/22172030), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Comparison of MAOB expression, HiF-1 alpha expression, & peroxide generation in response to a MAOB substrate in normal human astrocytes & glioma cellsHiF-1 alpha (i, red), MAOB (ii, green), & superimposed HiF-1 alpha /MAOB signals with blue DAPI labeling (iii) of NHAs (A) & GBM cells (B). (C) Relative expression of HiF-1 alpha (white bars) & MAOB (black bars) in NHAs & in three GBM low-passage cultures, n = 6, mean ± SD. (D) MAOB inhibited (gray bars), endogenous (white bars) & 4-FBA-stimulated (black bars) reactive oxygen species (hydrogen peroxide) generation, shown as DCF per cell, in NHA & the three GBM cultures, n = 16, mean ± SD. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.6582), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha is activated in VSMCs during Ang II-induced vascular remodeling.WT mice were infused with saline or 1000 ng/kg/min Ang II for 28 days. a Immunofluorescence analysis of representative cross-sections of mice aortas for HIF1 alpha (red) & alpha -SMA (green), nuclei was stained with DAPI. VSMCs were isolated form WT mice & treated with 1 μM Ang II for 24 h. bHif1a mRNA was measured by qPCR analysis. c HIF1 alpha protein was detected by western blot. **P < 0.01, ***P < 0.001, n = 3 per group, statistical significance was determined by the unpaired t-test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31320613), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effects of one- & five-dosage dry needling (1D, 5D) on iNOS, HIF-1 alpha , COX-2, & VEGF expressions in a needling-treated muscle. (a) Representative Western blot images. The quantification of the protein levels for (b) iNOS, (c) HIF-1 alpha , (d) COX-2, & (e) VEGF. Values are expressed as mean ± SD. *Indicates the significant difference (P < 0.05) between the sham groups (s1D & s5D). #Represents the significant difference (P < 0.05) between the 1D & 5D groups. DN: dry needling. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23346198), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of ENMD-1198 on tumor vascularization & tumor cell proliferation. A) Immunohistochemical analysis of tumor vascularization (CD31-positive vessels). Densitometric analysis of images from CD31-stained tissue sections from all tumors showed that vessel area was significantly reduced in ENMD-1198-treated tumors (*P < 0.01). B) ENMD-1198 significantly decreased the number of proliferating (BrdUrd-positive) tumor cells in tissue sections (*P < 0.01). For both vessel area & cell proliferation, representative images are illustrated. Results shown are the mean ± SEM. C) Western blot analysis for HIF-1 alpha expression in HCC tumors showed a substantial reduction of HIF-1 alpha in ENMD-1198 treated tumors. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/18651980), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Paraffin: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Enhanced expression of glycolytic genes in livers of DEN-Wwox delta Hep mice.a mRNA expression levels of HIF1 alpha glycolytic target genes in DEN-treated control & Wwox delta Hep (cKO) mice at the age of 1 a, 3 b, 6 c & 10 d months (n = 3 for each group). e Immunohistochemical staining of HIF1 alpha , PKM2, & GLUT1 in 6 & 10 months of DEN-treated Wwox delta Hep & control liver tissues. Images were taken at × 40 magnification (Bar = 100μm). f ChIP experiment with HIF1 alpha antibody on DEN-treated control & Wwox delta Hep (cKO) mice followed by qRT-PCR analysis of HIF1 alpha target genes. * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Error bars indicate ± SEM Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41419-018-0510-4), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Chromatin Immunoprecipitation: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Involvement of CBP in BNIP3 expression regulated by HIF-1 alpha & FOXO3 under hypoxia. (A) UCB-hMSCs were incubated with hypoxia condition for 24 h. Co-immunoprecipitation of HIF-1 alpha & FOXO3 with IgG & CBP were shown in left panel. IgG was used as a negative control. The total protein expressions of HIF-1 alpha , FOXO3, CBP & beta -actin in lysate were shown in right panel. n = 3. (B) CBP (20 μM) was pretreated to UCB-hMSCs, & cells were incubated with hypoxia for 24 h. The BNIP3 mRNA expression level was analyzed by qPCR. n = 6. (C) BNIP3 & beta -actin protein expressions were analyzed by western blot. Data represent mean ± S.E. n = 4. (D, E) Sample DNA was immuno-precipitated with RNA polymerase, IgG, HIF-1 alpha & FOXO3 specific antibodies. CHIP (top panel) & lysate (bottom panel) samples were amplified with the primers of GAPDH & BNIP3 promoters. Quantitative CHIP data was analyzed by qPCR, & shown in the right panel. n = 4. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Quantitative data are presented as a mean ± S.E.M. All blot images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Lack of HIF-P4H-1 increases the amount of p53. (a) Western blot analysis of p53 in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (b,c) Western blot analysis of p53 in Hif-p4h-1 & scrambled (Scrm) siRNA transfected HEK293 cells (b) & wt MEFs treated with 50 µM FG4497 for 24 h (c). (d) qPCR analysis of p53 mRNA in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (e) Analysis of p53 protein turnover rate. Hif-p4h-1−/− & wt MEFs were treated with 200 μg/ml of cycloheximide for the indicated time points & whole cell lysates were blotted for p53. (f) Western blot analysis of MDM2 in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (g) Western blot analysis of p53 & MDM2 in wt MEFs treated with 10 μM nutlin-3a for 24 h with or without overexpression of human HIF-P4H-1 (OE). (h,i) Western blot analysis of HIF1 alpha in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h (h) & in scrambled & Hif-p4h-1 siRNA transfected HEK293 cells (i). (j) Western blot analysis of ubiquitination of p53 in Hif-p4h-1 & scrambled siRNA transfected HEK293 cells. The cells were transfected with HA-ubiquitin & endogenous p53 was immunoprecipitated followed by Western blotting with anti-HA & anti-P53 antibodies. (k) Western blot analysis of p53 in Hif-p4h-1−/− & wt MEFs were treated with or without 10 μM MG132 for 5 h. Data are presented as representative Western blots & as mean ± s.d., n = at least 3 individual MEF isolates or experiments. *P < 0.05, **P < 0.01 & ***P < 0.001, two-tailed Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig. 5. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-17376-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha -mediated repression of ENT1 expression in Caki-1 cells under hypoxia. Comparison of HIF-1 alpha & HIF-2 alpha protein expression in human matched renal normal-tumor samples (n=11) (A) & 3 RCC cell lines (786-O, 769-P, Caki-1) under normoxia or hypoxia for 6 h (B). (C) Schematic diagram of two hypoxia response elements (HRE) in the promoter of ENT1. (D) CHIP-qPCR analysis of HIF-1 alpha at the promoter of ENT1 in Caki-1 cells, exposed under normoxia or hypoxia for 48 h. Two different HIF-1 alpha antibodies (610958, BD Biosciences; NB100-105, Novus) were used to pull down HIF-1 alpha . The protein expression level of HIF-1 alpha (E) & mRNA expression level of SLC29A1 (F) in Caki-1 under normoxia or hypoxia & HIF-1 alpha knockout-Caki-1 cells. (G) The expression level of ARNT1 (HIF-1 beta ) was detected in Caki-1, transfected with ARNT1 specific siRNAs or NC. Student's t test (two-tailed) was used. (H, I) The expression level of ENT1 in Caki-1, transfected with ARNT1 specific siRNAs or NC, under normoxia or hypoxia. Oneway-ANOVA analysis was used. The results are expressed as mean ± SEM (n=3). ***P<0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32206108), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - L. donovani infection induces HIF-1 alpha expression in CD11chi splenic DCs in an IRF-5 dependent manner.Mice were infected with 2x107 amastigotes intravenously. (A) Real-time PCR analysis of HIF-1 alpha mRNA expression in CD11c+ cells purified from C57BL/6 mice at various time points after infection. (B) Immunoblot analysis of HIF-1 alpha expression in CD11c+ cells from C57BL/6 mice (upper panel) & densitometric analysis normalized to beta -actin expression & expressed as fold increase to results obtained with naïve mice (lower panel). (C) Real-time PCR analysis of HIF-1 alpha expression in sorted CD11c+ cells from Irf-5flox/floxCre- & Irf-5flox/floxCMV-Cre+. (D) Immunoblot analysis of Hif-1 alpha expression in CD11c+ cells population of Irf5flox/floxCre- (left upper panel) & Irf-5flox/floxCMV-Cre+ (right upper panel), & densitometric analysis normalized to beta -actin expression & expressed as fold increase to results obtained with naïve mice (lower panels). (E) Real-time PCR analysis of Hif-1 alpha expression in CD11c- splenocytes from Irf-5flox/floxCre- & Irf-5flox/floxCMV-Cre+. (F) Real-time PCR analysis of HIF-1 alpha mRNA expression in BMDC from Irf-5flox/floxCMV-Cre+ & Cre- mice. All data represent mean ± SEM combined from 3 independent experiments. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1004938), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Optical redox ratio (ORR) demonstrates reduced glycolytic phenotype following 24 hours of YC-1 treatment. ORR of A549 & A549RR cells before, 12 & 24 hours following 50 μM YC-1 treatment to inhibit HIF-1 (a,b). Immunoblotting for HIF-1 alpha (c) & PDK-1 (d) before & 24 hours following YC-1 treatment to verify in of HIF-1 alpha . Representative immunoblots were horizontally cropped at the molecular weight indicated (e). Uncropped exposures can be seen in Supplemental Fig. 1. Experiments were performed in triplicate across 3 independent experiments. *p < 0.05 compared to A549 0 hr control, †p < 0.05 compared to A549RR 0hrs. Scale bar in images represents 50 μm. ME – Main effect of cell type & time; however, no significant interactions were found between the two main effects. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-27262-y), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - A beta facilitates HIF1 alpha synthesis & autophagy inhibition via mTOR activation. (A) SK-N-MC cells exposed to A beta (5 μM) for 0–48 h. HIF1 alpha & beta -actin expression analyzed by WB. n = 3. (B) Cells pretreated w/ NAC (1 mM) for 30 min prior to A beta treatment for 24 h. HIF1 alpha & beta -actin expression analyzed by WB. n = 3. (C,E) Cells incubated w/ rapamycin (10 nM) for 30 min prior to A beta treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) & 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1 alpha & beta -actin analyzed by WB. n = 6. (D) Protein samples immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples blotted w/ 4EBP1 & eIF4E-specific antibodies. n = 3. (F) Cells exposed to PF4708671 (10 μM) for 30 min prior to A beta treatment for 24 h. HIF1 alpha & beta -actin expression detected by WB. n = 6. (G) Cells exposed to cycloheximide (4 μM) for 30 min prior to A beta treatment for 24 h. HIF1 alpha & beta -actin expressions detected by WB. n = 6. (H) Cells pretreated w/ rapamycin (10 nM) for 30 min, incubated w/ A beta for 24 h & analyzed by WB w/ LC3, p62 & beta -actin specific antibodies. n = 3–6. (I) LC3 puncta visualized by confocal microscopy. Presented results merged images. Green & red fluorescents indicate LC3 & PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells pretreated w/ trehalose (10 μM) for 30 min prior to A beta treatment for 24 h. Cytotoxicity measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability measured by trypan blue exclusion assay. Data presented as a mean ± SE. n = 6. Each blot image presented as representative image. *p < 0.05 vs. control, #p < 0.05 vs. A beta treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28790888), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - FDFT1 inhibits glycolysis through suppressing AKT-mTOR-HIF1 alpha pathway in CRC.aFDFT1 overexpression reduced glucose uptake in CT26 cells. bFDFT1 overexpression decreased lactate production via glycolysis in CT26 cells. c ECAR reduced when FDFT1 overexpressed in CT26 cells. d OCR increased when FDFT1 overexpressed in CT26 cells. eFDFT1 knockdown increased glucose uptake in CT26 cells. fFDFT1 knockdown increased lactate production via glycolysis in CT26 cells. g ECAR increased when FDFT1 knocked down in CT26 cells. h OCR decreased when FDFT1 knocked down in CT26 cells. i, mFDFT1 overexpression inhibited protein & mRNA expression of mTOR-targeted glycolytic enzymes, including GLUT1, HK2, PGK1, GPI, & LDHA, in CT26 cells. j, mFDFT1 knockdown increased protein & mRNA expression of mTOR-targeted glycolytic enzymes, including GLUT1, HK2, PGK1, GPI, & LDHA, in CT26 cells. k, nFDFT1 overexpression decreased protein & mRNA expression of AKT, mTOR, & HIF1 alpha . l, nFDFT1 knockdown increased protein & mRNA expression of AKT, mTOR, & HIF1 alpha . o Photograph of dissected tumors (the first line: normal diet, second line: FMD + glucose, third line: FMD, n = 5). p The tumor volumes measured every 2 days after 13th day. The FMD + glucose group can reverse tumor growth inhibition induced by FMD (n = 5; ns: P = 0.1838; P = 0.0001). q The protein level of FDFT1 & mTOR in dissected tumor samples from normal diet group, FMD group & FMD + glucose group measured by western blotting. r The glucose level in these three groups. Error bars, mean ± SD, data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared w/ control group (or normal diet group). #P < 0.05, ##P < 0.01. Image collected & cropped by CiteAb from following publication (//pubmed.ncbi.nlm.nih.gov/32313017), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - WNT11 is induced by hypoxia or hypoxic mimetics in different cell types.(A) Increased Wnt11 mRNA in EMSC adipocytes (Day 12) after hypoxia-mimetic treatments. EMSC adipocytes were treated with CoCl2 (0.1 mM), DFO (0.1 mM) or DMOG (0.1 mM) for 24 hrs. Values were normalized to Tbp mRNA & are expressed relative to control (n = 3). (B,C) Increased Wnt11 mRNA by hypoxia in EMSC preadipocytes & adipocytes (Day 0–12 after differentiation) (B), & C2C12 myoblast & myocyte (Day 0 & 8 after differentiation) (C). Wnt11 mRNA was assessed by quantitative PCR in cells exposed to air (21% O2) or hypoxia (1% O2) for 24 hrs. (n = 4). Values were normalized to Tbp mRNA & are expressed relative to 21% O2 samples (left panel). (D) Immunoblot analyses of HeLa cells under normal air or hypoxia for 24 hrs. (E,F) Induction of Wnt11 by increasing concentrations of DMOG in MDA-MB-231 cells (E) & 4T1 cells (F). (G) EMSCs treated with 0.1 mM DMOG for the indicated times. Wnt11 & Vegf mRNA expression was measured by qPCR & normalized to Tbp mRNA (n = 4). (H) WNT11 protein levels after DMOG treatment normalized to alpha -Tubulin (upper panel; n = 4). Representative immunoblots of EMSCs treated with 0.1 mM DMOG for the indicated times (Lower panel). (I) Protein expression in MDA-MB-231 cells treated with 0.1 mM DMOG. (J) Induction of Wnt11 promoter activity by hypoxia or hypoxia mimetics. pGL3-Wnt11 promoter plasmid was transfected into C2C12 cells. Cells were incubated with DMOG (left panel, n = 4) or under 21% O2 or 1% O2 (right panel, n = 8) for 24 hrs. For panels (A–C,G,H,J), values are mean ± s.e.m. *p < 0.05, **p < 0.01. For panels of immunoblotting, laminin, alpha -tubulin, & ERK were used as loading controls, WNT11 normalized to alpha -Tubulin was shown. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Ccl7 is a HIF1 alpha direct target gene.a qPCR analysis of Ccl7 mRNA expression in Hif1afl/fl & Hif1a delta SMC VSMCs treated with vehicle, CoCl2, normoxia, or hypoxia (2% O2) for 6, 12, & 24 h. *P < 0.05, **P < 0.01, ***P < 0.001, n = 6 (independent experiments) per group. VSMCs isolated from Hif1afl/fl mice & Hif1a delta SMC mice were infected with oxygen-stable HIF1 alpha -expressing lentivirus, & then treated with Ang II for 24 h, bCcl7 mRNA was measured by qPCR & c CCL7 protein was detected by ELISA. d qPCR analysis of Ccl7 mRNA expression in vehicle or Ang II-treated Hif2afl/fl & Hif2a delta SMC VSMCs. e Schematic diagram of the mouse Ccl7 promoter illustrating the HREs in the regulatory region; the upstream regions were numbered in relation to the transcription initiation site. f Luciferase-reporter constructs under the control of the mouse Ccl7 promoter. HEK293T human embryonic kidney cells transiently transfected with the luciferase construct, & cotransfected with empty vector or HIF1a expression plasmids. Standard dual-luciferase assays were performed. EV, empty vector. **P < 0.01, n = 3. g, h ChIP assays of vehicle or Ang II-treated wild-type VSMCs using HIF1 alpha or HIF2 alpha antibodies. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. i, j ChIP assays of vehicle or Ang II-treated Hif1afl/fl & Hif1a delta SMC VSMCs using HIF1 alpha antibody. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. Statistical significance was determined by one-way ANOVA test followed by the unpaired t-test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31320613), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Hypoxic/necrotic core in GemOE tumors enhances Ac-HMGB1 secretion(A–C) Adjacent sections from a GemOE tumor IHC stained for geminin (A), c-Abl (B) & HMGB1 (C). (D) The level of circulating HMGB1 measured using specific ELISA assay performed on serum isolated from samples collected 7 weeks after mice were injected in mammary fat pads with naïve HME cells (n = 10, no tumors developed) or GemOE cells (n = 30, tumor-bearing, p = 0.00042). Two different sets (E, G, I & K) & (F, H, J & L) of adjacent sections from GemOE orthotopic mammary tumors stained with H & E (E & F), or IHC stained for geminin (G & H), HMGB1 (I & J) as well as HIF-1 alpha (K) or hypoxyprobe (L). N denotes necrosis within these tumors that are shown adjacent to the hypoxic cells as indicated by high HIF-1 alpha or hypoxyprobe staining. These cells are also expressing cytoplasmic HMGB1. (M) The levels of HMGB1 detected using specific ELISA assay released from iGem9, iG197, iG240 or iG257 cells grown under normoxic (N) or hypoxic (H) conditions. Experiments were done in triplicates 3 different times, **represents p < 0.001. (N) The level of acetylated HMGB1 passively diffused from naïve HME, iG197, iG240 or iG257 cells after repeated freeze & thaw cycles. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26989079), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - A beta induces the expression of cell cycle regulatory proteins. (A) SK-N-MC cells were exposed to A beta (5 μM) for 24 h. The mRNA expression levels of CDK5, P35 & P39 were analyzed by real-time PCR. The mRNA expression level was normalized by beta -actin mRNA expression level. Data represent the mean ± SE. n = 4. (B)hif1 alpha specific- & non-targeting (NT) siRNA were transfected to the cells for 24 h prior to A beta treatment. Cyclin D1, CDK4, cyclin E, CDK2, HIF1 alpha & beta -actin was detected by western blot. n = 3. (C–F) Cells were pretreated with trehalose (10 μM), rapamycin (10 nM), PF4708671 (10 μM) & cycloheximide (4 μM) for 30 min prior to A beta treatment for 24 h. Cyclin D1, CDK4, cyclin E, CDK2 & beta -actin were detected by western blot. n = 3–6. (G) Mouse hippocampal neurons were transfected with hif1 alpha specific- & NT siRNAs for 24 h prior to A beta treatment for 24 h. Samples were blotted with Cyclin D1, CDK4, cyclin E, CDK2 & beta -actin specific antibodies. n = 3–6. (H) Mouse hippocampal neurons were pretreated with trehalose (10 μM) for 30 min & incubated with A beta for 24 h. cyclin D1, CDK4, cyclin E, CDK2, HIF1 alpha & beta -actin were analyzed by western blot. n = 3–6. Data are presented as a mean ± SE. *p < 0.05 vs. control, #p < 0.05 vs. A beta treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28790888), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Hypoxia induces expression of WNT11 through VHL.(A,B) Higher basal levels of WNT11 protein in Vhl-deleted cells (lenti-Cre infected Vhlf/f). EMSCs isolated from Vhlf/f mouse were infected with lentivirus carrying either GFP gene (for control) or Cre recombinase (for knockout). Non-infected cells were also used as a control. Immunoblot analysis of control or Vhl KO EMSCs treated with 0.1 mM DMOG (A), & EMSCs exposed to air (21% O2) or hypoxia (1% O2) for 24 hrs (B). Laminin, alpha -tubulin, & lamin A/C were used as loading controls, WNT11 normalized to alpha -Tubulin was shown. (C,D) Inactivation of the Vhl gene results in increased Wnt11 mRNA. Wnt11 & Vegf mRNA levels in liver (C) or duodenum (D) were measured by qPCR in Liver-VhlcKO or duodenum-VhlcKO & control mice (n = 5 per group). Values normalized to Tbp mRNA are expressed relative to tissues from control mice. For panels (C,D), values are mean ± s.e.m. *p < 0.05, **p < 0.01. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - QSCs are hypoxic in the niche & express HIF2A, but not HIF1A.(A) Timeline of in vivo pimonidazole labeling in SC-INTACT mice & representative confocal images of uninjured/resting EDL myofibers (n >50 myofibers from n = 3 mice) showing that nmGFP+ QSCs were pimonidazole+. Scale bars: 50 μm & 10 μm (insets). Inset images show that pimonidazole signals were relatively enriched in the cytoplasm of QSCs. Arrowheads indicate a QSC; asterisks indicate a myonucleus. (B) Percentage of pimonidazole+ QSCs. (C) Timeline of in vivo CCI-103F labeling in C57BL/6 mice & representative images of uninjured/resting EDL myofibers (n >50 myofibers from 3 mice) showing that nmGFP+ QSCs were CCI-103F+. Arrowheads indicate a QSC; asterisks indicate a myonucleus. Scale bar: 20 μm. (D) Percentage of CCI-103F+ QSCs. (E & F) Representative images of uninjured/resting EDL myofibers from C57BL/6 mice (n >50 myofibers from 6 mice/group) showing that most QSCs were HIF2A+, but HIF1A–. Scale bars: 10 μm. (G) Percentage of HIF1A+ & HIF2A+ QSCs. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Role of HIF-1 alpha , FOXO3 in hypoxia-induced BNIP3 expression. (A) UCB-hMSCs were incubated with various durations of hypoxia (0–48 h). The protein expressions of HIF-1 alpha & beta -actin were detected by western blot. n = 4. (B) UCB-hMSCs were pretreated with NAC (5 mM) for 30 min prior to hypoxia incubation for 24 h. The protein expressions of HIF-1 alpha , lamin A/C & beta -tubulin in non-nuclear & nuclear fractionized cell samples were assessed by using western blot. n = 3. (C) UCB-hMSCs were immuno-stained with HIF-1 alpha & PI (magnification ×600). Scale bars, 37.5 µm. (D) HIF1A siRNA or NT siRNA was transfected to cells prior to hypoxia treatment for 24 h. The mRNA expression of BNIP3 was analyzed by qPCR. n = 6. (E) The protein expressions of BNIP3 & HIF-1 alpha were detected by western blot. n = 4. (F) NAC (5 mM) was pretreated to UCB-hMSCs prior to hypoxia treatment for 24 h. FOXO3, lamin A/C & beta -tubulin proteins expressions were assessed by western blot. n = 3. (G) FOXO3 siRNA transfected to UCB-hMSCs prior to hypoxia treatment for 24 h. The FOXO3 mRNA expression was measured by qPCR. n = 6. (H) BNIP3, FOXO3 & beta -actin expressions were detected by western blot. n = 3. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Lamin A/C & beta -tubulin were used as nuclear & non-nuclear protein controls, respectively. Quantitative data are presented as a mean ± S.E.M. All blots & confocal images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - ROS overproduction mediated apoptosis induced by combined treatment of DDP & HIF-1 alpha in PC-3 xenografts & cell culture. (a) ROS monitored by DHE (red) & nuclei by DAPI (blue) staining in PCa xenografts (scale bars, 50 µm). (b) MDA formation of PCa xenografts was examined after various treatments (c,d). PC-3 cells were treated with DDP, si-HIF-1 alpha plasmid, or both, in the presence or absence of NAC (5 mM) or DHLA (0.25 mM) for 24 h. Total lysates (c) & culture media (d) were used to detect cellular H2O2 level. (e,f) Western analysis for HIF-1 alpha as well as cleaved caspase-3 & PARP in PC-3 cells following various treatments. Data were presented as mean ± SD of three independent experiments. *p < 0.05 versus control group; #p < 0.05 versus si-HIF-1 alpha or DDP group; $p < 0.05 versus DDP/si-HIF-1 alpha group. The original blots are presented in Supplementary Figure 8. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-07973-4), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - miR-128 downmodulation regulates MAFG increase in hypoxic condition both in vitro & in vivo. C2C12 cells exposed to hypoxia (2% O2) for 4 h, & miR-128 levels (a) determined by RT-qPCR on total RNAs after normalization w/ the small RNU6. Data are expressed as relative to values obtained in untreated control which set equal to 1. Each column in the panels represents the mean ± SD of 3 independent experiments. ∗p < 0.05. Western blotting analysis (b) of HIF-1 alpha , MAFG, & HMOX-1 performed on total extracts from the same cells, as described in Materials & Methods. Tubulin used as a loading control. mRNA levels of HMOX-1 & x-CT genes (c) determined by RT-qPCR on total RNAs, & relative changes calculated by comparing treated cells versus control, after normalization w/ c-ABL. Data reported as relative to values obtained in normoxia, which set equal to 1. Each column in the panel represents the mean ± SD of 3 independent experiments. ∗p < 0.05. Representative laser Doppler analysis (d) of blood flow before & 4 hours after (post 4 h) hindlimb ischemia procedure (n = 3-4 mice/group). Representative Western blot analysis & densitometric analysis (e) of MAFG & HMOX-1 protein levels performed on hindlimb muscle lysates from littermates after sham procedure (sham) or not-ischemic limb (NIL) & ischemic limb (IL) 4 h after femoral artieriectomy. Tubulin protein levels used as loading controls (∗p < 0.05 versus sham; n = 3-4 hindlimb/group). miR-128 levels (f) determined by RT-qPCR on total RNAs after normalization w/ the small RNU6. The values obtained in not-ischemic limb (NIL) set equal to 1. The data are expressed as the mean ± standard error & are representative of 3 independent experiments. ∗p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29138682), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Effect of ENMD-1198 on HIF-1 alpha & VEGF expression in HCC cells. To determine effects of ENMD-1198 on nuclear HIF-1 alpha expression, HUH-7 cells were incubated for 16 h ± ENMD-1198 & subsequently exposed to hypoxia (1% O2, 4 h). A) Western blot analysis of whole protein showed that ENMD-1198 effectively blunted hypoxic induction of HIF-1 alpha protein. B) In addition, treatment with ENMD-1198 down-regulated constitutive VEGF-A mRNA levels in HCC cells (HepG2), as measured by real-time PCR (* P < 0.01) (n = 3/group). VEGF-A mRNA expression is normalized to beta -actin. C) ELISA analysis for VEGF in culture supernatants (HepG2). Hypoxia (20 h, 1% O2) markedly increased VEGF protein. ENMD-1198 treatment lowered VEGF secretion under hypoxic conditions (*P < 0.05). D) Western blot analysis for VEGF. Cells were incubated under either non-hypoxic, or hypoxic conditions ± ENMD-1198. Treatment with ENMD-1198 slightly lowered the hypoxic induction of VEGF, as determined by densitometry. Bars: SEM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/18651980), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Upregulation of HIF-1 alpha in human PCa. (a) HIF-1 alpha protein was detected by western blot in nonmalignant (RWPE-1 & BPH1) & PCa cell lines (PC-3, DU145, LNCaP, & 22RV1) as indicated. (b–d) Total RNA extracted from RWPE-1, BPH1, PC-3, DU145, LNCaP, & 22RV1 cells was subjected to qRT-PCR for HIF-1 alpha (b), VEGF (c) & GLUT4 (d). (e) The HIF-1 alpha promoter-driven reporter (firefly luciferase) & a control vector (Renilla luciferase) were co-transfected into RWPE-1, BPH1, PC-3, DU145, LNCaP, & 22RV1 cells for measurement of luciferase activity. HIF-1 alpha promoter activity was calculated as a ratio of firefly to Renilla activity. (f) Human normal & malignant tissue (Gleason score 9) sections were probed with HIF-1 alpha antibody (scale bars, 100 µm). (g) Protein expression of HIF-1 alpha , VEGF, & GLUT4 were examined with western blot, in PC-3, DU145, & LNCaP cells after various treatments as indicated. Data are expressed as mean ± SD of seven independent experiments. $p < 0.05 versus RWPE-1 or BPH1 cells or normal tissue. *p < 0.05 versus control group. #p < 0.05 versus si-HIF-1 alpha or DDP group. Original blots are shown in Supplementary Figure 5. C: Ctrl; D: DDP; S: si-HIF-1 alpha ; D/S: DDP/si-HIF-1 alpha . Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-07973-4), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Cx43-overexpressing cells regulate the expression of HIF-1 alpha , vascular endothelial growth factor (VEGF), & the proliferation of endothelial cells. (a) The expression of Cx43 & HIF-1 alpha was measured in Cx43-overexpressing cells by Western blot analysis. beta -actin expression served as loading controls for & total protein. Inserted values indicated relative proteins expression in comparison with beta -actin; (b) Cx43-overexpressing cells were cotransfected with pCLNCX-6× HRELuc & pTCYLacZ plasmids. At 6 h post-transfection, their luciferase activities were determined & normalized with beta -gal activity. Data shown were the mean ± SD (n = 4); (c) The conditioned medium of Cx43-overexpressing cells was measured by ELISA. Data shown were the mean ± SD (n = 4); & (d) The conditioned medium of Cx43-overexpressing cells reduced the proliferation of endothelial cells. The HEMC-1 cells treated with conditioned medium of Cx43-overexpressing cells were examined the proliferation activity. Cell viability was assessed by the WST-1 assay. Data shown were the mean ± SD (n = 4). * p < 0.05; *** p < 0.001. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/16/1/439), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Prolonged hypoxia increases glutamine metabolism in PDAC cells, & HIF‐2 alpha can promote non‐canonical glutamine metabolism in chronic hypoxic conditions. (A) Time course of glutamine consumption at 1%, 3% or 20% O2, each time data point is an average of triplicate experiments. (B) Panc‐1 & Capan‐2 were incubated for 48 hrs at 1%, 3% or 20% O2, GLS1, GOT1 & GOT2 protein were measured by Western blot. beta ‐Actin was used as loading control. (C) Panc‐1 & Capan‐2 were incubated for 48 hrs at 1%, 3% or 20% O2, GLS1, GOT1 & GOT2 mRNA were measured by qRT‐PCR. Data are presented as mean ± S.D. from three independent experiments. (D & F) Si‐HIF‐2 alpha ‐transfected Panc‐1 & Capan‐2 cultured at 1% or 3%O2 for 48 hrs. The level of HIF‐2 alpha & glutamine metabolism enzymes mRNA & protein were determined by qRT‐PCR (mRNA) & Western blot (protein), & beta ‐actin was used as loading control. Data are presented as mean ± S.D. from three independent experiments. *P < 0.05, **P < 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28544376), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha & PFKFB3 are protective against hIAPP mediated beta -cell death. a Representative immunoblotting (left) & quantification (right) of the whole cell extract (WCE) from asynchronous INS 832/13 cells treated with either HIF1 alpha inhibitor KC7F2 (10 μM) or PFKFB3 siRNA (75 nM) for 56 h versus control. Cells were transduced with hIAPP adenovirus (75 MOI) or LacZ adenovirus as control (75 MOI) for last 32 h of culture. DMSO or CTRL siRNA, respectively were added to LacZ or hIAPP to equal DMSO or PFKFB3 siRNA used in experimental groups. GAPDH was used as a loading control. Data are presented as mean ± SEM, n = 4, *p < 0.05, **p < 0.01. b Quantification of DNA content distribution after FACS analysis of INS 832/13 cells treated as described in (a). Data are the mean ± SEM, n = 4, *p < 0.05, **p < 0.01. c The frequency of TUNEL positive INS 832/13 cells treated as described in (a). Data are the mean ± SEM, n = 4 in (a) & (b), & n = 3 independent experiments in (c). Statistical significance was analyzed by one-way ANOVA test with Tukey’s post-test (*p < 0.05, **p < 0.01). See Supplementary Fig. 10 for additional supportive data Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31213603), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Immunohistochemical & Western blot analysis of HIF-1 alpha & VEGF expression in OVA-challenged mice.(A & B) Immunostaining for HIF-1 alpha & VEGF in nasal mucosa following the last challenge. Positive HIF-1 alpha signals are brown & are predominantly nuclear & VEGF is predominantly cytoplasmic (magnification 400×, scale bar = 20 µm). (C & D) Representative Western blot analysis showing HIF-1 alpha & VEGF (with beta -actin as a loading control) expression in the nasal mucosa 24 h after the last challenge. All densitometric analyses are presented as the relative ratio of each molecule to beta -actin & the ratio in negative control mice was set to 100. Values represent means±SEM (n = 6 mice). *Significantly different from negative control, p<0.05; #significantly different from positive control, p<0.05. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0048618), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - DC101, evofosfamide, & HIF-1 alpha shRNA treatment of HT1080 sarcomasA. Western blot of HT1080 cells stably transduced with HIF-1 alpha shRNA (sh.HIF-1 alpha ) or scrambled control shRNA (sh.Scr) & grown in 21% oxygen or 1% oxygen. beta -actin serves as loading control. B. Growth of HT1080 cells stably transduced with sh.HIF-1 alpha or sh.Scr in athymic nude mice. Groups were treated with control IgG 20 mg/kg 3 times per week, DC101 20 mg/kg 3 times per week, and/or evofosfamide (Evo) 50 mg/kg 5 times per week. C. Photos of H&E staining, cleaved caspase 3 IF, cleaved caspase 3 & CD31 IF, CD31 IHC, pimonidazole IHC, & HIF-1 alpha IHC. D. Graphs of microvessel density, pimondizale area, & nuclear HIF-1a expression. Bars represent standard deviation. **p < 0.05 compared to all other groups. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.10212), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - CAPE inhibits MCT-induced HIF-1 alpha expression & PDGF-BB production in rats. (A) Rats were treated with PBS or CAPE (5 or 10 mg/kg) from days 14–28 after MCT treatment 14 days (60 mg/kg). Preparation of lung tissues was analyzed by Western blot to determine the levels of HIF-1 alpha protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used as a loading control. (B) Serum levels of PDGF-BB in rat was assayed by an ELISA kit. (C) Representative images of immunohistochemical staining for HIF-1 alpha in lung sections from the rats. Scale bars, 50 μm. (D) Immunofluorescence staining of alpha-smooth muscle actin ( alpha -SMA) & HIF-1 alpha in lung sections from rats. Scale bars, 50 μm. Data in (A,B) are expressed as mean ± SEM of three independent experiments. *** p < 0.01, as compared with the PBS group. ### p < 0.01, as compared with the rats exposed to MCT alone. Results are representative of three rats per experimental group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30909527), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - mTOR expression is inversely correlated with FDFT1 expression.aFDFT1 overexpression inhibited the protein level of mTOR, whereas FDFT1 knockdown increased the protein level of mTOR in CT26 & SW620 cells. bFDFT1 expression & mTOR expression were negatively correlated in CRC patient samples. Scale bar: 200 µm. c The mTOR silencing efficiency of the siRNA in CT26 & SW620 cells was validated by western blotting. d The effect of mTOR silencing on FDFT1 expression level in CT26 was evaluated by western blotting. e The protein level of mTOR when FDFT1 knockdown combined with or without mTOR inhibitor in CT26. f The protein level of pS6k, S6k, pS6, & S6 under the effect of fasting & FDFT1 overexpression in CRC cells. g, h CCK8 proliferation assays showed that the silencing of mTOR decreased the proliferation of CT26 & SW620 cells. k, n The silencing of mTOR reduced glucose uptake & lactate production in CT26 & SW620 cells. i, j, l, o The silencing of mTOR decreased the ECAR & increased the OCR in CT26 & SW620 cells. m The silencing of mTOR decreased the expression of AKT, HIF1 alpha , & proteins encoded by relevant glycolytic genes, such as GLUT1, HK2, LDHA, GPI, PGK1, in CT26 & SW620 cells. p–t Based on TCGA data set analysis, mTOR expression was positively correlated with AKT1, HIF1 alpha , GLUT1, HK2, & LDHA expression. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32313017), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Chromatin Immunoprecipitation: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha -mediated repression of ENT1 expression in Caki-1 cells under hypoxia. Comparison of HIF-1 alpha & HIF-2 alpha protein expression in human matched renal normal-tumor samples (n=11) (A) & 3 RCC cell lines (786-O, 769-P, Caki-1) under normoxia or hypoxia for 6 h (B). (C) Schematic diagram of two hypoxia response elements (HRE) in the promoter of ENT1. (D) CHIP-qPCR analysis of HIF-1 alpha at the promoter of ENT1 in Caki-1 cells, exposed under normoxia or hypoxia for 48 h. Two different HIF-1 alpha antibodies (610958, BD Biosciences; NB100-105, Novus) were used to pull down HIF-1 alpha . The protein expression level of HIF-1 alpha (E) & mRNA expression level of SLC29A1 (F) in Caki-1 under normoxia or hypoxia & HIF-1 alpha knockout-Caki-1 cells. (G) The expression level of ARNT1 (HIF-1 beta ) was detected in Caki-1, transfected with ARNT1 specific siRNAs or NC. Student's t test (two-tailed) was used. (H, I) The expression level of ENT1 in Caki-1, transfected with ARNT1 specific siRNAs or NC, under normoxia or hypoxia. Oneway-ANOVA analysis was used. The results are expressed as mean ± SEM (n=3). ***P<0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32206108), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Upregulation of HIF-1 alpha in the spleen restricts CD8+ T cell expansion.(A-B) Mice were infected with 2x107 amastigotes intravenously & euthanized at various time points after infection. HIF-1 alpha expression in splenocytes was assessed by real-time PCR (A) & immunoblot analysis (B). (C-F) 2x104 OT-I CD8+ T cells were adoptively transferred into recipient mice a day prior to infection with ovalbumin-transgenic (PINK) L. donovani amastigotes. (C) Graphs represent the average number of OT-I CD8+ T cells found in the spleen from Hif1aflox/flox & Hif1a+/-mice at d7 & d14 p.i.. (D) Percentage of OT-I CD8+ T cells at d7 & d14 p.i.. (E) Percentage of gated OT-I CD8+ expressing CD62Llo/int & negative for CD127. (F) Percentage of OT-I CD8+ T cells that were CD44+ CD127+CD62Lhi. All data represent mean ± SEM of one of 3 independent experiments, n = 5. * denotes p<0.05 & ** denotes p<0.01. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1004938), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - CAPE inhibits MCT-induced HIF-1 alpha expression & PDGF-BB production in rats. (A) Rats were treated with PBS or CAPE (5 or 10 mg/kg) from days 14–28 after MCT treatment 14 days (60 mg/kg). Preparation of lung tissues was analyzed by Western blot to determine the levels of HIF-1 alpha protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used as a loading control. (B) Serum levels of PDGF-BB in rat was assayed by an ELISA kit. (C) Representative images of immunohistochemical staining for HIF-1 alpha in lung sections from the rats. Scale bars, 50 μm. (D) Immunofluorescence staining of alpha-smooth muscle actin ( alpha -SMA) & HIF-1 alpha in lung sections from rats. Scale bars, 50 μm. Data in (A,B) are expressed as mean ± SEM of three independent experiments. *** p < 0.01, as compared with the PBS group. ### p < 0.01, as compared with the rats exposed to MCT alone. Results are representative of three rats per experimental group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30909527), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Role of HIF-1 alpha , FOXO3 in hypoxia-induced BNIP3 expression. (A) UCB-hMSCs were incubated with various durations of hypoxia (0–48 h). The protein expressions of HIF-1 alpha & beta -actin were detected by western blot. n = 4. (B) UCB-hMSCs were pretreated with NAC (5 mM) for 30 min prior to hypoxia incubation for 24 h. The protein expressions of HIF-1 alpha , lamin A/C & beta -tubulin in non-nuclear & nuclear fractionized cell samples were assessed by using western blot. n = 3. (C) UCB-hMSCs were immuno-stained with HIF-1 alpha & PI (magnification ×600). Scale bars, 37.5 µm. (D) HIF1A siRNA or NT siRNA was transfected to cells prior to hypoxia treatment for 24 h. The mRNA expression of BNIP3 was analyzed by qPCR. n = 6. (E) The protein expressions of BNIP3 & HIF-1 alpha were detected by western blot. n = 4. (F) NAC (5 mM) was pretreated to UCB-hMSCs prior to hypoxia treatment for 24 h. FOXO3, lamin A/C & beta -tubulin proteins expressions were assessed by western blot. n = 3. (G) FOXO3 siRNA transfected to UCB-hMSCs prior to hypoxia treatment for 24 h. The FOXO3 mRNA expression was measured by qPCR. n = 6. (H) BNIP3, FOXO3 & beta -actin expressions were detected by western blot. n = 3. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Lamin A/C & beta -tubulin were used as nuclear & non-nuclear protein controls, respectively. Quantitative data are presented as a mean ± S.E.M. All blots & confocal images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Cx43-knockdown cells regulate the expression of HIF-1 alpha , c-Src, VEGF & the proliferation of endothelial cells. (a) The expression of Cx43, c-Src, & HIF-1 alpha was measured in Cx43-knocknown cells by Western blot analysis. beta -actin expression served as loading controls for & total protein. Inserted values indicated relative protein expression in comparison with beta -actin; (b) Cx43-knockdown cells were cotransfected with pCLNCX-6× HRELuc & pTCYLacZ plasmids. Then, cells were treated with 100 ng/μL PP2 or DMSO for 1 h. At 6 h post-transfection, their luciferase activities were determined & normalized with beta -gal activity. Data shown were the mean ± SD (n = 4); (c) The conditioned medium of Cx43-knockdown cells was measured by ELISA. Data shown were the mean ± SD (n = 4); (d) The conditioned medium of Cx43-knockdown cells reduced the proliferation of endothelial cells. The HEMC-1 cells treated with conditioned medium of Cx43-knockdown cells were examined for proliferation activity. Cell viability was assessed by the WST-1 assay. Data shown were the mean ± SD (n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/16/1/439), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Involvement of CBP in BNIP3 expression regulated by HIF-1 alpha & FOXO3 under hypoxia. (A) UCB-hMSCs were incubated with hypoxia condition for 24 h. Co-immunoprecipitation of HIF-1 alpha & FOXO3 with IgG & CBP were shown in left panel. IgG was used as a negative control. The total protein expressions of HIF-1 alpha , FOXO3, CBP & beta -actin in lysate were shown in right panel. n = 3. (B) CBP (20 μM) was pretreated to UCB-hMSCs, & cells were incubated with hypoxia for 24 h. The BNIP3 mRNA expression level was analyzed by qPCR. n = 6. (C) BNIP3 & beta -actin protein expressions were analyzed by western blot. Data represent mean ± S.E. n = 4. (D, E) Sample DNA was immuno-precipitated with RNA polymerase, IgG, HIF-1 alpha & FOXO3 specific antibodies. CHIP (top panel) & lysate (bottom panel) samples were amplified with the primers of GAPDH & BNIP3 promoters. Quantitative CHIP data was analyzed by qPCR, & shown in the right panel. n = 4. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Quantitative data are presented as a mean ± S.E.M. All blot images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Precision quantitative image analysis workflow for the characterization of functional ITH.(a) Representative areas with positive immunohistochemical (IHC) staining for HIF-1 alpha , HIF-2 alpha , phospho-mTOR S2448, phospho-S6RP S235/236 & Ki-67. Only nuclear HIF-1 alpha & HIF-2 alpha expression was included in the analysis. Scale bar, 100 μm. (b) Overview of the quantitative IHC analysis process using a representative image of a whole-slide scanned pT1M1 tumour after phospho-mTOR S2448 staining. The image was overlaid with a virtual grid consisting of 1 mm2 squares that defines the tumour regions to be analysed. Non-cancerous tissue was subtracted manually (dark grey) & a positive pixel count was performed on the corrected area. Positivity per square was expressed as percentage of positive pixels. If a square contained non-tumorous tissue, the positivity was normalized to the corrected square area. Computed values were depicted as a heat map. Coloured squares represent tumour, whereas open squares indicate completely non-tumorous areas. Squares defined as peripheral tumour zone are marked by a black line. Scale bar, 1 cm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27291893), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Regulation of OA development by HIF-1 alpha . Plain radiographs of the entire bodies at 8 weeks of age (A), & knee joints 8 weeks after OA surgery (B) of Cntl & cKO littermates. Scale bars, 10 mm & 1 mm, respectively. (C) Safranin O & TUNEL staining’s of Cntl & KO knee joints at 8 weeks after OA surgery (n = 9 & 11, respectively). Tamoxifen induction was performed at 7 weeks. Inset boxes in top images indicate the regions of high magnification Safranin O & TUNEL images. Inset boxes in middle images indicate the regions of immunofluorescence in (G). White dotted lines in TUNEL images indicate the cartilage area. Scale bars, 400 µm & 100 µm, respectively. (D) OARSI (Osteoarthritis Research Society International) score of OA development. Lines show the mean ± SD. *P < 0.05. (E) Rate of TUNEL-positive cells in the cartilage area. Bars show the mean ± SD. *P < 0.05. (F) mRNA levels of Hif1a in articular cartilage tissues of Cntl & KO. Bars show the mean ± SD. *P < 0.05. (G) Immunofluorescence of Hif1a, Mmp13, Hif2a, I kappa B alpha , Ser 32 & 36 dual phosphorylated I kappa B alpha , Rela, & C1qtnf3. Scale bars, 20 µm. The percentage of positive cells in the immunofluorescence is shown below. *P < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32214220), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - TSA reduces HIF-1 alpha & VEGF expression & up-regulates the expression of PEDF.Real-time PCR (A) & Western blot assays (B) were performed on RPE cells treated with 0–0.5 μM TSA & 150 μM CoCl2. (A) Cells were treated with 0–0.5 μM TSA for 14 h & then co-treated with 150 μM CoCl2 for 6 h for the analysis of gene expression by real-time PCR. Changes in HIF-1 alpha mRNA levels were not statistically significant. CoCl2 causes a fourfold enhancement of VEGF mRNA expression; but at 0.5 μM TSA, the mRNA level of VEGF reduces to less than half of that in cells treated with CoCl2 only. TSA induces a statistically significant increase in the mRNA level of PEDF. (B) Cells were treated with 0–0.5 μM TSA for 18 h & then co-treated with 150 μM CoCl2 for 6 h for Western blot analysis. TSA reduces the CoCl2-induced HIF-1 alpha & VEGF protein levels by 4.3-fold & 5.7-fold, respectively, & up-regulates PEDF protein level by threefold. (C) Densitometry data for Western blot of HIF-1 alpha , VEGF & PEDF. (*: t test p<0.05; **: t test p<0.01). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0120587), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Role of HIF-1 alpha , FOXO3 in hypoxia-induced BNIP3 expression. (A) UCB-hMSCs were incubated with various durations of hypoxia (0–48 h). The protein expressions of HIF-1 alpha & beta -actin were detected by western blot. n = 4. (B) UCB-hMSCs were pretreated with NAC (5 mM) for 30 min prior to hypoxia incubation for 24 h. The protein expressions of HIF-1 alpha , lamin A/C & beta -tubulin in non-nuclear & nuclear fractionized cell samples were assessed by using western blot. n = 3. (C) UCB-hMSCs were immuno-stained with HIF-1 alpha & PI (magnification ×600). Scale bars, 37.5 µm. (D) HIF1A siRNA or NT siRNA was transfected to cells prior to hypoxia treatment for 24 h. The mRNA expression of BNIP3 was analyzed by qPCR. n = 6. (E) The protein expressions of BNIP3 & HIF-1 alpha were detected by western blot. n = 4. (F) NAC (5 mM) was pretreated to UCB-hMSCs prior to hypoxia treatment for 24 h. FOXO3, lamin A/C & beta -tubulin proteins expressions were assessed by western blot. n = 3. (G) FOXO3 siRNA transfected to UCB-hMSCs prior to hypoxia treatment for 24 h. The FOXO3 mRNA expression was measured by qPCR. n = 6. (H) BNIP3, FOXO3 & beta -actin expressions were detected by western blot. n = 3. Western blot data were normalized by beta -actin, & qPCR data were normalized by ACTB mRNA expression level. Lamin A/C & beta -tubulin were used as nuclear & non-nuclear protein controls, respectively. Quantitative data are presented as a mean ± S.E.M. All blots & confocal images are representative. *p < 0.05 versus control, #p < 0.05 versus hypoxia. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213231717303804), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1 alpha protein is suppressed by ED71 but not by 1,25(OH)2D3.(A) Western analysis of Raw264.7 cells cultured in hypoxic conditions with or without 10−7 M of ED71 or 1,25(OH)2D3 (1,25D). (B) Hif1 alpha mRNA levels in Raw264.7 cells cultured in hypoxic conditions were analyzed by realtime PCR in the presence or absence of 10−7 M ED71 or 1,25(OH)2D3. Data represent mean Hif1 alpha expression relative to that of Actb ± SD (n = 5). (C) Levels of VDR transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean VDR expression relative to that of Actb ± SD (n = 5). (D) Western analysis of control (shControl) or VDR-suppressed (shVDR#1 or shVDR#2) Raw264.7 transformants cultured in hypoxic conditions with ED71 or 1,25(OH)2D3 (1,25D), both at 10−7 M. (E) M-CSF-dependent Ctsk Cre/Hifflox/flox cells were cultured in normoxic conditions to suppress HIF1 alpha in the presence of M-CSF (50 ng/ml) plus RANKL (25 ng/ml) with either ED71 or 1,25(OH)2D3 (1,25D) both at 10−7M for 4 days. Expression of Ctsk & NFATc1 was then assessed by realtime PCR. Data represent mean Ctsk or NFATc1 expression relative to that of Actb ± SD (n = 5). *P<0.05; **P<0.01; ***P<0.001. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0111845), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - NDRG1 was regulated by HIF‐1/2 alpha . (A‐E) The protein expression of the shown genes was detected by Western blot, respectively; beta ‐actin was used as the loading control. (A) RCC4 & 786‐O cells (B) were infected with VHL vector (VHL) or empty vector (EV). (C) Caki‐1 cells were transfected with VHL shRNAs (shVHL) or non‐specific control (NC). (D) RCC4 cells were infected with HIF‐1 alpha siRNAs (siHIF‐1 alpha ) or non‐specific control (NC). (E) 786‐O cells were transfected with HIF‐2 alpha shRNAs (shHIF‐2 alpha ) or non‐specific control (NC). (F) Representative images of HIF‐1 alpha , HIF‐2 alpha & NDRG1 protein expression from the IHC staining of human clear cell renal cell cancer specimens. Patient I: The protein levels of HIF‐1 alpha , HIF‐2 alpha & NDRG1 were low. Patient II: The protein levels of HIF‐1 alpha , HIF‐2 alpha & NDRG1 were medium. Patient III: The protein levels of HIF‐1 alpha , HIF‐2 alpha & NDRG1 were high (scale bar, 50 μm). (G & H) The protein level of NDRG1 is significantly correlated with HIF‐1 alpha (G) & HIF‐2 alpha (H). The protein levels were evaluated according to IHC scores, representing very low (score 0‐2), low (score 3‐4), high (score 5‐8) & strong (score 9‐12). The subjects were divided into four groups according to the IHC scores of HIFs in the tumours. (I) The protein level of NDRG1 is significantly correlated with the average of HIF‐1 alpha & HIF‐2 alpha in ccRCC tissues. The protein levels are evaluated according to IHC scores, representing very low (score < 3), low (score < 5), high (score < 8) & strong (score 8‐12). The subjects were divided into four groups according to the IHC scores of the average of HIF‐1 alpha & HIF‐2 alpha . Significance p‐values & r values according to Spearman's rank correlation Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32537867), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Bv, Ca, Fe, Ma, Pm, Pm, Rb, Sh, Xp

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Bv, Ca, Fe, Ma, Pm, Pm, Rb, Sh, Xp

Applications:

, , , , , , , , , , , , , , ,

Applications: , , , , , , , , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-105AF350
Alexa Fluor 405	NB100-105AF405
Alexa Fluor 488	NB100-105AF488
Alexa Fluor 532	NB100-105AF532
Alexa Fluor 594	NB100-105AF594
Alexa Fluor 647	NB100-105AF647
Alexa Fluor 700	NB100-105AF700
Alexa Fluor 750	NB100-105AF750
Alexa Fluor Plus 405	NB100-105AFP405
Alexa Fluor Plus 488	NB100-105AFP488
Alexa Fluor Plus 555	NB100-105AFP555
Alexa Fluor Plus 594	NB100-105AFP594
Alexa Fluor Plus 647	NB100-105AFP647
Alexa Fluor Plus 680	NB100-105AFP680
Allophycocyanin	NB100-105APC
Biotin	NB100-105B
CoraFluor 1	NB100-105CL1
DyLight 350	NB100-105UV
DyLight 405	NB100-105V
DyLight 488	NB100-105G
DyLight 550	NB100-105R
DyLight 594	NB100-105DL594
DyLight 650	NB100-105C
DyLight 680	NB100-105FR
DyLight 755	NB100-105IR
FITC	NB100-105F
HRP	NB100-105H
Janelia Fluor 525	NB100-105JF525
Janelia Fluor 549	NB100-105JF549
Janelia Fluor 646	NB100-105JF646
Janelia Fluor 669	NB100-105JF669
PE	NB100-105PE
PerCP	NB100-105PCP
mFluor Violet 450 SE	NB100-105MFV450
mFluor Violet 500 SE	NB100-105MFV500
mFluor Violet 610 SE	NB100-105MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Azide and BSA Free	NBP2-80760

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NB100-122

HIF-2 alpha/EPAS1 Antibody - BSA Free

(37 Reviews)

(876 Publications)

0.1 ml

NB100-122

<b>Orthogonal Strategies Validation. </b>Formalin-fixed paraffin-embedded tissue sections of human placenta were probed for HIF-2 alpha/EPAS1 mRNA (ACD RNAScope Probe, catalog #410598; Fast Red chromogen, ACD catalog # 322750). Adjacent tissue section was processed for immunohistochemistry using Rabbit Polyclonal (Novus Biologicals catalog # NB100-122) at 1:100 dilution with one-hour incubation at room temperature followed by incubation with anti-rabbit IgG VisUCyte HRP Polymer Antibody (Catalog # VC003) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). Specific staining was localized to trophoblastic cells.

HIF-2-alpha-EPAS1-Antibody-Immunohistochemistry-NB100-122-img0028.jpg

Image shows a specific band for HIF-2 alpha in 0.5 mg/mL of hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

<b>Genetic Strategies Validation. </b>HIF-2-alpha-EPAS1-Antibody-Knockout-Validated-NB100-122-img0035.jpg

<b>Biological Strategies Validation. </b>HIF-2 alpha in human retinal and choroidal primary endothelia lysates using .. Image from verified customer review.

<b>Biological Strategies Validation. </b>HIF-2 alpha in MDA-MB-231 cell lysate (overexpression and endogenous samples) using . did not react to HIF-1 alpha overexpression. Image from verified customer review.

<b>Biological Strategies Validation. </b>HIF-2-alpha-EPAS1-Antibody-Western-Blot-NB100-122-img0030.jpg

MDA-MB-231 cells were exposed to 20% or 1% O2 for 16 hours, and chromatin immunoprecipitation (ChIP) was performed with the indicated antibody (Ab). Primers flanking the HIF binding site were used for qPCR. ChIP image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>Lane 1: Cobalt chloride treated COS7 nuclear extracts. Lane 2: Untreated COS7 nuclear extracts.

786-O cells without or with VHL overexpression. Image from verified customer review.

<b>Biological Strategies Validation. </b>Analysis using the HRP conjugate of NB100-122. Detection of normoxic and hypoxic nuclear rat cell lysates.

HIF-2 alpha immunoreactivity in human cardiac myocytes stained with NB100-122.

Expression and correlation of 3 antibodies: HIF-1 alpha, HIF-2 alpha and HAF in ccRCC. (A, C & E) Positive nuclear staining to the 3 antibodies: HIF-1 alpha, HIF-2 alpha and HAF, in primary ccRCCs at high magnification (100X). (B, D & F) Heterogenous nuclear staining of 3 antibodies in a tumor at low magnification (20X). (G, H and I). Correlation of 3 antibodies with the Fuhrman grade.Citation: Ambrosetti D, Dufies M, Dadone B, Durand M, Borchiellini D, Amiel J, et al. (2018) The two glycolytic markers GLUT1 and MCT1 correlate with tumor grade and survival in clear-cell renal cell carcinoma. PLoS ONE 13(2): e0193477. //doi.org/10.1371/journal.pone.0193477

<b>Genetic Strategies Validation. </b>Reduction of HIF-2alpha levels leads to protection in UV-triggered apoptosis, but not for apoptosis caused by glucose and serum starvation in 786-O cells. Parental 786-O or those either stably expressing wild-type VHLp19 or stably infected with a control vector (pSuperRetro) or a pool of two HIF-2alpha shRNAs vectors [21] were grown to confluence and lysed. Cell alpha-tubulin.

Sp1 and HIFs contribute to the synergistic activation of multiple genes in OVSAYO cells under SSH.b) Activation of multiple genes under hypoxia is dependent on HIFs. Western blotting is also shown for HIFs. Data shown are the mean (n = 3) ± SD.

Western Blot: Rabbit Polyclonal HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis of HIF-2 alpha/EPAS1 antibody on mouse liver nuclear extracts. Image from a verified customer review.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - Muscle repair following eccentric contraction–induced injury is concomitant with dynamic alterations of HIF2A & HIF1A expression in SCs.(A–C) Representative images of EDL myofibers from injured muscles at various time points (n >50 myofibers from 3 mice/group/time point) & stained for Pax7, DAPI, & EdU (A), HIF2A (B), or HIF1A (C). Scale bars: 20 μm. Arrowheads indicate SCs. (D) Number of Pax7+ SCs per myofiber at various time points. (E) Percentage of EdU+ SCs at various time points. (F) Percentage of HIF2A+ SCs at various time points. (G) Percentage of HIF1A+ SCs at various time points. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - Genetic ablation of HIF2A in QSCs leads to transient activation, proliferation, & differentiation of SCs.(B) Representative images of myofibers from SC-HIF2AKO mice & control littermates (n >50 myofibers from 5 mice/group; 10 dpr). Immunofluorescence of Pax7 (red), HIF2A (green), MyoD (purple), & DAPI (blue) staining revealed HIF2A–MyoD+ & HIF2A+MyoD– SCs (arrowheads) in SC-HIF2AKO & control mice, respectively. Scale bar: 10 μm. Image collected & cropped by CiteAb from following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - EpEX & EpCAM activate a STAT3-HIF2 alpha signal for EpEX/EpCAM-mediated iPSC formation.(A) iPSCs were infected with two different EpCAM shRNAs (two clones, #1 & #2). The protein expressions of EpCAM & HIF2 alpha were detected by Western blotting. (B) MEFs were stimulated by EpEX (1 μg/mL) at the indicated times. Nuclear-translocation was detected with a specific antibody against HIF2 alpha (n = 3). (C) Immunofluorescence staining was performed to detect subcellular localization of HIF2 alpha . Nuclei were stained with DAPI. Scale bar: 10 μm. (D) MEFs were treated with STAT3 inhibitor (WP1066, 10 μM), & then stimulated with EpEX for 30 min. The nuclear-translocation of HIF2 alpha was detected by Western blotting with anti-HIF2 alpha antibody (n = 3). (E) iPSC morphology was observed at day 20 after induction. Reprogramming of Oct4-GFP MEFs was induced by transfection of OSKM, OE + EpEX, & KE + EpEX with or without STAT3 inhibitor WP1066, or HIF2 alpha shRNA (n = 3). Scale bar: 50 μm. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep41852), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Gel Super Shift Assays: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - Manipulation of DNA methylation at HRE sequences alters HIF binding. (A) The Hypoxia Response Element (HRE) contains a CpG site that can be methylated which prevents HIF1 & HIF2 binding in vitro. EMSA of in vitro–translated HIF1 & HIF2 binding to 32P-labelled unmethylated & methylated HRE probes. Competition with 250X molar excess of unlabelled/unmethylated HRE probe (lanes 3, 7,12, 16) or unlabelled control HRE-free probe (lanes 4, 8, 13, 17). HIF1 & HIF2 complexes bound to unmethylated HRE or methylated HRE supershifted with anti-HIF1 alpha (lanes 9 & 18) & anti-HIF2 alpha (lanes 5 & 14). (B) RCC4-VHL cells were grown in normoxia (NT) or treated with 25 µM decitabine (5aza) or grown in hypoxia (1%) for 48 hrs & with the addition of 25 µM decitabine (1% + 5aza). QPCR was performed using primers specific to VEGF, uPAR, TGF alpha , GUS, U1AsnRNP1. Samples were normalised to relative expression of the housekeeping gene, ACTIN. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-21524-5), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - HIF-1 alpha /2 alpha expression in myeloid-specific KO mice targeting the HIF pathway. (A) Images of the colon of wild-type (WT), myeloid-specific Hif-1a KO (hMRP8 Hif-1a KO) or von Hippel Lindau (Vhl) KO (hMRP8 Vhl KO) mice, immunostained for MRP8 (green) & the DNA-binding regions of Hif-1a mRNA (red). Mice were fed with 5% DSS for 4 days prior to immunostaining analyses. Note that there were no MRP8-positive cells that were positive for Hif-1a mRNA in hMRP8 Hif-1a KO (middle column) mice, but we observed many cells that were double positive for MRP8 & Hif-1a mRNA in hMRP8 Vhl KO mice (right column). (B) Images of the colon of hMRP8 Vhl KO mice fed with 5% DSS as in A, immunostained for MRP8 (green) & HIF-1 alpha (red, upper row) or HIF-2 alpha (red, bottom row). DAPI-stained nuclei are shown in blue. White boxes in A & B indicate the regions magnified in the lower or right images, respectively. Yellow arrowheads in A & B indicate cells positive for both markers. Scale bars: 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29967068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - HIF2 alpha stabilization results in exocrine cell atrophy & expansion of duct-like tubular structures. (A) Body weight (left panel), pancreas weight (middle panel) & body/pancreas weight ratio (right panel) in Pdx1-Cre;HIF2dPA & control mice at 2 & 8 weeks of age. Data are presented as mean ± SD. (B) HIF2 alpha accumulation in Pdx1-Cre;HIF2dPA analyzed by Western blot with anti-HA antibody. Two independent two-week-old control & mutant mice are shown. beta -actin protein was used for loading control. Full-length blots are presented in Supplementary Fig. 2. (D) Immunofluorescence analysis of HIF2 alpha in two-week-old control pancreata. Endogenous HIF2 alpha expression is observed in islets (marked by an white asterisk) but not in exocrine tissue. (D) Robust HIF2 alpha accumulation in the pancreas of two-week-old Pdx1-Cre;HIF2dPA mice. Hematoxylin/Eosin-stained pancreatic sections from P0 (E,F), two- (I,J) & eight-week-old (M,N) Pdx1-Cre;HIF2dPA & control mice. Inset in N shows an area with adipose tissue in Pdx1-Cre;HIF2dPA pancreata. Immunofluorescence of amylase & KRT19 shows no differences between Pdx1-Cre;HIF2dPA & control mice at P0 (G,H). Duct-like tubular structures & loss of amylase immunoreactivity in two- (K,L) & eight-week-old (O,P) Pdx1-Cre;HIF2dPA mice compared to control mice. Note areas with normal acini in 8-week-old Pdx1-Cre;HIF2dPA mice (white asterisk in O). Insets in (H,L & P) show higher magnification pictures. DAPI staining is shown in blue in (C,D,G,H,K,L,O & P). Scale bars = 50 µm for (C,D); 100 µm for (E–P). ***P < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30209343), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody - BSA Free [NB100-122] - QSCs are hypoxic in the niche & express HIF2A, but not HIF1A.(A) Timeline of in vivo pimonidazole labeling in SC-INTACT mice & representative confocal images of uninjured/resting EDL myofibers (n >50 myofibers from n = 3 mice) showing that nmGFP+ QSCs were pimonidazole+. Scale bars: 50 μm & 10 μm (insets). Inset images show that pimonidazole signals were relatively enriched in the cytoplasm of QSCs. Arrowheads indicate a QSC; asterisks indicate a myonucleus. (B) Percentage of pimonidazole+ QSCs. (C) Timeline of in vivo CCI-103F labeling in C57BL/6 mice & representative images of uninjured/resting EDL myofibers (n >50 myofibers from 3 mice) showing that nmGFP+ QSCs were CCI-103F+. Arrowheads indicate a QSC; asterisks indicate a myonucleus. Scale bar: 20 μm. (D) Percentage of CCI-103F+ QSCs. (E & F) Representative images of uninjured/resting EDL myofibers from C57BL/6 mice (n >50 myofibers from 6 mice/group) showing that most QSCs were HIF2A+, but HIF1A–. Scale bars: 10 μm. (G) Percentage of HIF1A+ & HIF2A+ QSCs. Data represent the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//www.jci.org/articles/view/96208), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Fi, Ha, Pm, Rb, Re, Sh

Species Glossary

Reactivity: Hu, Mu, Rt, Fi, Ha, Pm, Rb, Re, Sh

Applications:

, , , , , , , , , , , , , ,

Applications: , , , , , , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-122AF350
Alexa Fluor 405	NB100-122AF405
Alexa Fluor 488	NB100-122AF488
Alexa Fluor 532	NB100-122AF532
Alexa Fluor 594	NB100-122AF594
Alexa Fluor 647	NB100-122AF647
Alexa Fluor 700	NB100-122AF700
Alexa Fluor 750	NB100-122AF750
Alexa Fluor Plus 405	NB100-122AFP405
Alexa Fluor Plus 488	NB100-122AFP488
Alexa Fluor Plus 555	NB100-122AFP555
Alexa Fluor Plus 594	NB100-122AFP594
Alexa Fluor Plus 647	NB100-122AFP647
Alexa Fluor Plus 680	NB100-122AFP680
Allophycocyanin	NB100-122APC
Allophycocyanin/Cy7	NB100-122APCCY7
Biotin	NB100-122B
CoraFluor 1	NB100-122CL1
DyLight 350	NB100-122UV
DyLight 405	NB100-122V
DyLight 488	NB100-122G
DyLight 550	NB100-122R
DyLight 594	NB100-122DL594
DyLight 650	NB100-122C
DyLight 680	NB100-122FR
DyLight 755	NB100-122IR
FITC	NB100-122F
HRP	NB100-122H
Janelia Fluor 525	NB100-122JF525
Janelia Fluor 549	NB100-122JF549
Janelia Fluor 646	NB100-122JF646
Janelia Fluor 669	NB100-122JF669
PE	NB100-122PE
PE/Atto594	NB100-122PEATT594
PE/Cy5.5	NB100-122PECY55
PE/Cy7	NB100-122PECY7
PerCP	NB100-122PCP
mFluor Violet 450 SE	NB100-122MFV450
mFluor Violet 500 SE	NB100-122MFV500
mFluor Violet 610 SE	NB100-122MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-122SS

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NB100-304

53BP1 Antibody - BSA Free

(14 Reviews)

(817 Publications)

0.1 mg

NB100-304

Irradiated cochlear spiral ganglion cells, mouse, frozen section of fixed material. Image from verified customer review.

<b>Genetic Strategies Validation. </b>53BP1 was detected in immersion fixed HeLa cells (left) but was not detected in 53BP1 knockout Hela cells (right) using Rabbit Anti-human 53BP1 polyclonal antibody (Catalog #NB100-304) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to nuclei.

Total protein from HeLa, A431, Neuro2A, and PC12 was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1.0 ug/mL in 5% block buffer and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. The observed molecular weight for these samples are ~250 kDa and the theoretical molecular weight is 214 kDa.

Human breast tumors stained with 53BP1 antibody. Image from verified customer review.

Detection of Human and Mouse 53BP1 by IHC. Sample: FFPE sections of human ovarian carcinoma (left) and mouse teratoma (right). Antibody: 53BP1 Antibody (Catalog #NB100-304) used at a dilution of 1:1000 (1ug/mL). Detection: DAB.

Human medulloblastoma (DAOY) and mouse astrocyte (C8D1A) cell lines were exposed for 48 hours to DMSO or 1ug/mL of the DNA damaging agent EPE. Cells were immunostained for 53BP1 (green). The nuclei were counterstained with DAPI (blue). Image from verified customer review.

An intracellular stain was performed on RH-30 cells with 53BP1 Antibody (Catalog #NB100-304AF647) (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.

Immunoprecipitation analysis lysates from HCC44 cells in 1% NP40. Image from verified customer review.

<b>Biological Strategies Validation. </b>53BP1-Antibody-Immunocytochemistry-Immunofluorescence-NB100-304-img0041.jpg

<b>Biological Strategies Validation. </b>53BP1-Antibody-Western-Blot-NB100-304-img0045.jpg

HeLa cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with 53BP1 Antibody conjugated to DyLight 550 (NB100-304R) at 5 ug/ml for 1 hour at room temperature. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

An intracellular stain was performed on Ntera2 cells with 53BP1 Antibody NB100-304 (blue) and a matched isotype control NBP2-24891 (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1.0 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550 (SA5-10033, Thermo Fisher).

Whole cell lysate from U2OS or 293T cells. Bands indicate an observed molecular weight of ~220 kDa and the theoretical molecular weight is 214 kDa.

<b>Biological Strategies Validation. </b>Upper Panel: 53BP1 foci in proliferating MEFs. Lower Panel: 53BP1 foci in proliferating MEFs exposed to 10 Gy of IR.

Embryonic Fibroblast cells pre-extraction for 5 mins with CSK buffer. Fixed with 4% PFA and 75% Ethanol. Primary Antibody at 1:1000. Secondary Antibody at 1:1000. Image from verified customer review.

<b>Biological Strategies Validation. </b>Upper Panel: Control untreated cells. Lower Panel: Cells exposed to irradiation (10 Gy) and probed for 53BP1 foci. Cells were grown on coverslips, fixed with 4% paraformaldehyde, methanol permeabilized, blocked for 1 h, RT. Incubated with primary antibody (1:200) overnight, washed 3x with PBS, probed with tubulin (Alexa Fluor 594) antibody for 2 h, RT. Washed 3x with PBS, mounted on slides using prolong gold, imaged using Nikon confocal microscope (100x oil). Image from verified customer review.

<b>Genetic Strategies Validation. </b>53BP1-Antibody-Immunocytochemistry-Immunofluorescence-NB100-304-img0044.jpg

<b>Biological Strategies Validation. </b>53BP1-Antibody-Immunocytochemistry-Immunofluorescence-NB100-304-img0047.jpg

53BP1-Antibody-Immunocytochemistry-Immunofluorescence-NB100-304-img0046.jpg

Ntera2 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-53BP1 Antibody NB100-304 at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:1000 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

NIH3T3 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-53BP1 Antibody NB100-304 at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:1000 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Staining of 53BP1 in human colon cancer using DAB with hematoxylin counterstain.

An intracellular stain was performed on A431 cells with 53BP1 Antibody (Catalog #NB100-304AF647) (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.

For 53BP1 staining, samples were incubated for 1 hour with 1:200 53BP1 primary antibody in PBS/BSA 1%, at room temperature. Green: 53BP Blue: DAPI. Image from verified customer review.

<b>Biological Strategies Validation. </b>DYNLL1 is required for 53BP1-dependent p53 responses to Nutlin-3. a Immunoblot analysis of the MCF-7 cell lines used in (a) with anti-53BP1 or anti-RPA32 antibodies prior to N3 treatment. b Quantification of n = 3 independent experiments represented in (a), each performed in triplicate. Mean ± SD. c Indicated parental or stably complemented 53BP1-/- MCF-7 cell lines were incubated in the presence (11 days) or absence (7 days) of Nutlin-3 (4 µM)

<b>Genetic Strategies Validation. </b>Depletion of PNUTS and NPAT induces a global DDR.(A) Induction of telomere dysfunction–induced foci (TIF) upon candidate knockdown in HeLa cells from Fig 5. Depletion of TRF2 was used as a positive control. 53BP1 was detected by indirect IF and telomeres by FISH with a Cy3-[CCCTAA]3 probe. (B) Frequency distribution of the number of TIFs per cell from (A). n indicates the number of cells analyzed for each condition and red lines mark the mean.

<b>Biological Strategies Validation. </b>HPV+ HNSCCs have decreased expression of NHEJ and HR proteins including DNA-Pk and BRCA2Cells were treated with mock or 4 Gy radiation, harvested and lysed at 15 minutes post-treatment, and analyzed by western blot for relative expression of indicated proteins. beta -actin was used as a loading control. Shown is a representative blot from 2 independent experiments.

<b>Biological Strategies Validation. </b>LNCaP cells in culture were treated for 24 hrs with DMSO or ENZ, then irradiated with 0 or 1 Gy.Cell were fixed and immunostained for 53BP1 0.5, 1.5 and 24 hrs after irradiation. Immunofluorescent images were obtained by confocal microscopy. White bar = 20 microns. A) DMSO alone, B) ENZ alone, C) 1 Gy, D) ENZ + 1 Gy. White bar = 20 microns.

<b>Left:</b> Simple Western lane view shows lysates of HepG2 human hepatocellular carcinoma cell line, loaded at 1 mg/ml. A specific band was detected for 53BP1 at approximately 353 kDa (as indicated) using both 4 µg/ml and 20 µg/ml of Rabbit Anti-Human 53BP1 Antigen Affinity-purified Polyclonal Antibody (Catalog # NB100-304) followed by HRP-conjugated Goat Anti-Rabbit Secondary Antibody (Catalog # 042-206). This experiment was conducted under reducing conditions and using the 66-440kDa separation system. <b>Right:</b> Simple Western electropherogram showing the same Rabbit Anti-Human 53BP1 Antigen Affinity-purified Polyclonal Antibody (Catalog # NB100-304) tested at 4 µg/ml (blue line) and 20 µg/ml (green line) in the HepG2 human hepatocellular carcinoma cell line.

Representative immunohistochemistry staining for 53BP1 expression in pancreatic adenocarcinoma. (A) is high intensity of 53BP1 expression and (B) is low intensity of 53BP1 expression.

53BP1 was detected in immersion fixed U-2 OS human osteosarcoma cell line using Rabbit anti-53BP1 Antigen Affinity Purified Polyclonal Antibody conjugated to DyLight 650 (Catalog # NB100-304C) (light blue) at 10 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - Depletion of TPX2 causes defects in 53BP1 ionizing radiation-induced foci formation.(A) The protein level of 53BP1 is not affected by miRNA-mediated depletion of TPX2. Levels of actin used as loading controls. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0110994), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Nuclear intensity of key BER proteins.A) Quantification of mean nuclear fluorescence intensity is shown for PARP1 (**** P < 0.0001, MDA-231 to MDA-157, HCC1806, & MDA-468), PAR (**** P < 0.0001, MDA-468 to MDA-157, MDA-231, & HCC1806), & p53 (**** P < 0.0001, MDA-157 to MDA-231, MDA-157 to MDA-468, MDA-157 to HCC1806, MDA-231 to MDA-468, MDA-231 to HCC1806, & MDA-468 to HCC1806). B) Representative fluorescent images used for quantification in A for PARP1, PAR, & p53 for all cell lines tested. Scale bar = 20 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31596905), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: 53BP1 Antibody - BSA Free [NB100-304] - Representative immunohistochemistry staining for 53BP1 expression in pancreatic adenocarcinoma. (A) is high intensity of 53BP1 expression & (B) is low intensity of 53BP1 expression. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23530749), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - H2A.J accumulates in the aging human epidermis.(a) H2A.J & Ki-67 co-localization in sections of human skin of the indicated ages by immunofluorescent staining. H2A.J showed mutually exclusive staining with the Ki-67 proliferation marker. (b) H2A.J & 53BP1 foci increase in aging human epidermis. Arrows indicate positively-stained nuclei. A quantification of positive cells for three biological replicates is shown below the immunofluorescence images, & the one-sided Mann–Whitney U-test was used to determine the statistical significance (*P<0.05, **P<0.01). Error bars represent mean±s.e. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms14995), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - 53BP1 cofractionates with lamins A/C in HDF. (A) HDF were treated with IR (5 Gy, 1 h) & fractionated by sequential treatments of detergent (CSK/T) & DNase I. Extracts were then processed for immunoblotting using the indicated antibodies. WCE, whole cell extract. P, pellet; S, supernatant. (B) HDF were treated with IR (2 Gy; 1 h) & extracted in situ with CSK/T followed by DNase I digestion before being processed for immunofluorescence using lamin A/C & 53BP1 antibodies. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25645366), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Depletion of Ajuba results in activation of the DNA damage response. (A) Staining for p53BP1 foci on cells fixed 72 h after transfection with siRNA #3, & siGFP as a negative control. Quantification of the number of p53BP1 foci on three independent siRNA experiments shown in (B). (C, D) Western blots for induction of Chk1 phosphorylation & induction of p53 phosphorylation, showed for the three target sites, 72 h after siRNA transfection. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23755068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Depletion of PNUTS & NPAT induces a global DDR.(A) Induction of telomere dysfunction–induced foci (TIF) upon candidate knockdown in HeLa cells from Fig 5. Depletion of TRF2 was used as a positive control. 53BP1 was detected by indirect IF & telomeres by FISH with a Cy3-[CCCTAA]3 probe. (B) Frequency distribution of the number of TIFs per cell from (A). n indicates the number of cells analyzed for each condition & red lines mark the mean. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30456372), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - HPV+ HNSCCs have decreased expression of NHEJ & HR proteins including DNA-Pk & BRCA2Cells were treated with mock or 4 Gy radiation, harvested & lysed at 15 minutes post-treatment, & analyzed by western blot for relative expression of indicated proteins. beta -actin was used as a loading control. Shown is a representative blot from 2 independent experiments. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26336991), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Spontaneous DNA breakage in G1-phase cells: GamGFP shows large spontaneous G1 53BP1 foci to contain multi-break clusters.The large spontaneous 53BP1 foci in undamaged cells, which occur solely in G1 (Harrigan et al., 2011;Lukas et al., 2011a), contain multiple DSBs that are marked by GamGFP. The GamGFP-53BP1 co-localization is more apparent in the absence of Ku. Data are from three (Ku80-proficient) or four (Ku80-defective) independent experiments of >25 cells each with Z-stacked (optically sectioned) nuclei.DOI://dx.doi.org/10.7554/eLife.01222.017 Image collected & cropped by CiteAb from the following publication (//elifesciences.org/articles/01222), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - 53BP1 cofractionates with lamins A/C in HDF. (A) HDF were treated with IR (5 Gy, 1 h) & fractionated by sequential treatments of detergent (CSK/T) & DNase I. Extracts were then processed for immunoblotting using the indicated antibodies. WCE, whole cell extract. P, pellet; S, supernatant. (B) HDF were treated with IR (2 Gy; 1 h) & extracted in situ with CSK/T followed by DNase I digestion before being processed for immunofluorescence using lamin A/C & 53BP1 antibodies. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25645366), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

$Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - 53BP1 focus formation. (A) Bars indicate the mean ± standard error of 53BP1 focus-forming cells in three independent control & three independent Asciz−/− primary MEF cultures under basal conditions (open bars), in response to 24-h aphidicolin treatment (black bars) & following 30 min recovery after ionizing radiation (grey bars). More detailed quantifications of foci/cell are shown in Supp. Fig. S2. (B) Subcelllular distribution of 53BP1 in cell fractionation assays. M601 is used as a mitochondrial marker, & tubulin as a cytosolic marker. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S1568786417301349), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - hsRAD51-II3A expressing cells accumulate 53BP1 foci after treatment with HU. Representative images depicting 53BP1 foci (green) in PCNA (red) positive cells after the indicated treatments. DNA is stained in blue. Scale bar = 10 μm. Dot plot depicts quantitation of 53BP1 foci after indicated treatments. Data is combined data consisting of at least 100 nuclei from two independent experiments. Red line represents the mean. Values are presented as mean ± SD. Statistical significance was determined using the Mann–Whitney U test. Source data are provided as a source data file Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31562309), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Persistent DNA damage exists in ALT cells. (A) IF-FISH showing DNA damage response (53BP1) in ALT (VA13, U2OS, SAOS2, SKLU-1), telomerase positive (MCF7, A549), & human normal fibroblast cells (MRC5, BJ). Scale bar: 10 μm. (B) Quantification of (A). Data represent the mean ± SEM, n = 3. More than 100 cells were counted for each experiment. (C) FACS analysis of apoptosis in cells listed in (A). (D) Quantification of (C). Data represent the mean ± SEM, n = 3. (E) Proliferation of p53-positive (VA13, U2OS) & p53-defictive (SAOS2, SKLU-1) ALT cells. (F) Western blot showing activation of JNK (phosphorylation) & accumulation of c-myc in ALT cells. Human normal BJ cells were used as a control Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31115790), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - LNCaP cells in culture were treated for 24 hrs with DMSO or ENZ, then irradiated with 0 or 1 Gy.Cell were fixed & immunostained for 53BP1 0.5, 1.5 & 24 hrs after irradiation. Immunofluorescent images were obtained by confocal microscopy. White bar = 20 microns. A) DMSO alone, B) ENZ alone, C) 1 Gy, D) ENZ + 1 Gy. White bar = 20 microns. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30933998), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - IMR90 cells depleted for Ajuba undergo a DNA damage response. (A,C,D) Western blots for Ajuba, Cyclin A, Rb, Chk1 phosphorylation, & PARP cleavage on lysates prepared from IMR90 cells 72 h after transfection with siRNA #3. The loading controls with GAPDH for each blot are shown. (B) Staining for p53BP1 in Ajuba-depleted cells (siRNA#3), with siGFP as a control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23755068), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - DYNLL1 is required for 53BP1-dependent p53 responses to Nutlin-3. a Immunoblot analysis of the MCF-7 cell lines used in (a) with anti-53BP1 or anti-RPA32 antibodies prior to N3 treatment. b Quantification of n = 3 independent experiments represented in (a), each performed in triplicate. Mean ± SD. c Indicated parental or stably complemented 53BP1-/- MCF-7 cell lines were incubated in the presence (11 days) or absence (7 days) of Nutlin-3 (4 µM) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30559443), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - RNA:DNA hybrids promote 53BP1 foci formation.(a) Immunofluorescence using the anti-flag & anti-53BP1 antibody in cells expressing Flag-tagged hybrid-binding (HB) domain or flag-tagged active RNaseH1. 53BP1 foci were quantified in cells expressing HB-domain or RNaseH1. Column bars represent the mean of n number of cells (described on each column), from three independent experiments. Error bars represent SEM. *P ≤ 0.05; **P < 0.01, ***P < 0.001 using the Kruskal-Wallis test followed by Dunn’s post hoc test. (b) Higher zoom representative images show HB-domain & 53BP1 co-localization, specifically in the euchromatin area. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30345028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - General chromatin effects of A∆rod & A∆rod∆hbs. (a) DNA staining (DAPI) in U2OS cells expressing A∆rod. Chromatin staining tended to be stronger on or around lamin A∆rod structures. (b) pRb is a known binding partner of lamin A, & so, it was co-stained as a positive control, revealing strong co-localization with the A∆ structures. (c) No co-localization between A∆rod structures & 53BP1 in HeLa cells indicates that chromatin is not damaged by the structures. (d) Similarly, gamma H2AX did not accumulate on A∆ structures. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/2073-4409/6/2/9), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - UFL1 regulates the ATM signaling. a U2OS cells expressing UFL1 tet-on shRNA1 were irradiated with 2 Gy IR. Thirty minutes later, cells were lysed & blotted with indicated antibodies. b Representative picture of gamma H2AX, 53BP1, & BRCA1 foci in control (Dox-) & UFL1 knockdown (Dox+) U2OS cells 1 h after 0.5 Gy treatment. Scale bars, 10 µm. c–e Quantification of intensities of gamma H2AX, 53BP1, & BRCA1 foci in control (Dox-) & UFL1 knockdown (Dox+) U2OS cells. Data presented as mean ± SD of n = 50 cells. **p < 0.01 by Student’s t-test. Source data are provided as a Source Data file Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30886146), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - The DNA damage response at telomeres uncapped through TRF2 depletion does not require BRCA1 or CtIPImmortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later & analysed by Western blotting as indicated. SMC1 & tubulin were used as loading controls. *non-specific band.Cells treated as in (A) were fixed 48 h after selection & stained with an anti-53BP1 antibody (green). Telomeres were visualized with a Cy3-conjugated (CCCTAA)3-PNA probe (red). Yellow arrowheads point to 53BP1 foci that co-localize with telomeres.Quantification of TIFs in cells treated as in (B). A minimum of 200 nuclei were scored for each sample. Error bars represent SD of two independent experiments. P-values were calculated using an unpaired two-tailed t-test. *P ≤ 0.05; NS, P > 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25582120), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - Hypoxia causes DNA damage in patient-derived colonosphere cultures in vitro(a) Two independent patient-derived colonospheres were cultured in normoxia (21%) or hypoxia (0.1%) for 24 hours. Immunofluorescence was then used to asses gamma H2AX (green) in the nuclei DAPI (blue). (b) Bar graphs showing the quantification of gamma H2AX observed in (a) (n=3), shown as percentage of the mean gamma H2AX intensity at normoxia. (c) Representative confocal pictures of the comet assay of colonospheres cultured as in (a). (d) Bar graphs showing the quantification of DNA damage observed with the comet assay (Extend tail moment) (n=73). (e) Colonospheres were cultured as in (a) & gamma H2AX was assessed by FACS. The plots show normoxic gamma H2AX levels in red & hypoxic gamma H2AX levels in blue in two independent cell lines. (f) Human colonospheres were exposed to hypoxia for the indicated periods of time. Cell lysates were then analyzed by Western blotting for the indicated markers (cropped). Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.21145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - TRF1HP1 alpha results in reduced TIFs induced by mutant hTR expression. Cells stably expressing TRF1HP1 alpha (WT or mutant variants of HP1 alpha ) were infected with lentivirus containing WT hTR, or mutant hTR (47A or TSQ1) on day 0, selected for stable expression after 48 h, & analyzed on day 5. a Fluorescence microscopy images of representative cells expressing mutant hTR 47A stained with telomeric (Tel-Cy3) peptide nucleic acid (PNA) probes (magenta in merged image) via fluorescent in situ hybridization (FISH), antibody against DNA-damage repair protein marker 53BP1 (green in merged image), & counterstained with DAPI. Zoom-in images (the last row) correspond to yellow-squared regions of the row above. b % TIF per telomere of each nucleus is quantified; n = 94–159 nuclei combining data of three independent experiments. ****p < 0.0001; n.s. (no significance). a, b Scale bar: 10 µm. c Upon TSQ1 expression, TRF1HP1 alpha results in fewer TIFs compared to Vonly, TRF1 or HP1 alpha controls. TRFHP1 alpha ~11.4% shows decreased TIFs compared to Vonly: ~19.7% ***p = 0.0008; TRF1: ~20.4% ***p = 0.0005; HP1 alpha : ~17.9% *p = 0.0137 (n = 30–38 nuclei). b, c Significance is assessed by one-way ANOVA & Dunnett’s multiple comparison test with 95% confidence level. d Fluorescence images of control cells overexpressing WT hTR (n = 27–36 nuclei). Same color scheme as a. TIFs quantification in the presence of e WT hTR or f Vonly (n = 28–36 nuclei) show minimal baseline DNA damage at telomeres. b, c; e, f Error bars represent s.e.m. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30181605), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Absence of Roma Leads to Activation of the DNA Damage Response & Genomic Instability(A) Representative immunofluorescence analysis of RPA2-pT21, gamma H2AX, & 53BP1 foci in tissue sections from WT & Roma−/− glands at 10dL. The white arrow indicates nucleolar gamma H2AX. Scale bars, 10 μm. The higher magnification panels on the right have been chosen to highlight the foci & are not zoomed images of the left panels.(B) Immunoblot analysis of cell-cycle checkpoint & DNA damage response in extracts from WT & Roma−/− glands at 10dL. Extracts from U2OS cells treated with 5mM HU were used as a damage control.(C) PCR analysis of p21Cip1 & MDM2 & quantitative real-time PCR analysis of p21Cip1 in extracts from 10dL WT & Roma−/− glands (p = 0.0007, Student’s t test).(D) Representative spectral karyotypes (top) & metaphase spreads (bottom panel) prepared from MECs isolated from WT & Roma−/− glands after a full natural wean.See also Figure S2. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27149840), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - A-T post-mitotic neurons are defective in DDR. In a, D30 post-mitotic neurons (D30) were tested by western blot with antibodies specific for the various ATM phosphosubstrates & cleaved PARP at various times after treatment with 5 Gy IR. beta -Actin was used as a loading control. DNA SSBs (b) & DSBs (c) were analyzed in neurons (D30) by alkaline & neutral comet assay, respectively, at two different times after treatment. 20 min after H2O2 or IR treatment was considered the time point with the maximum DNA damage. In b & c, representative photos of comets from untreated cells & at different time points following treatment. The ratio between treated & untreated tail moments of at least 50–70 cells per experimental point is shown in the graphs (values in %). One representative experiment out of three is shown. In d & e, formation & resolution of IR-induced nuclear foci. Neurons (D30) were irradiated with 0.5 Gy, collected at the indicated times & labeled for gamma -H2AX (d) & 53BP1 (e). For each treatment, the number of foci was scored from 100 cell nuclei per duplicate preparations & from three independent experiments (mean±S.D.) (d & e, bottom) For each time point, the difference between Ctrl & A-T was statistically significant (#P<0.01) (analysis performed by the Student's t-test) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25032865), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Exposure to radiation induces persistent genomic instability.(a) Time course of micronucleus frequency in binucleated cells exposed to 1Gy X-rays or Fe ions (average of two independent irradiations, error bars represent SEM). Paired Student's t-test, *p<0.05, **p<0.01. (b) Time course of gamma H2AX- 53BP1 positive foci per cell distribution in a population of 50 cells per condition following exposure to 1Gy of the indicated radiation. One of two experiments is shown. Chi-Square test NI vs. X-rays or Fe ions. day 7 & day 14 p<0.001. Day 21 p>0.05. (c) Immunofluorescence staining of gamma H2AX-53BP1 positive foci 7 days following exposure to 1Gy of the indicated radiation type. Scale bar is 10 µm. (d) Immunofluorescence staining for BrdU & 53BP1 7 days following exposure to 1Gy Fe ions. Scale bar is 10 µm. The graph represents the average of scoring in 5 different fields a total of 70 cells. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0108234), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - SPOC1 is recruited to I-SceI-induced DSBs where it colocalizes with 53BP1. (A) In situ immunofluorescence studies with U2OS19 ptight13 GFP-LacR cells with a stably integrated I-SceI cleavage site flanked by lac operator repeats reveals localization of the GFP-lac repressor protein (GFP-LacR) at the lac-operator DNA sequences in the nucleus before (−I-SceI) & 16 h after I-SceI-induced (+ I-SceI) DSB. In contrast, SPOC1 (white) & 53BP1 (red) are distributed throughout the nucleus in the absence of I-Scel, & partially colocalized in naturally occurring repair foci. After I-SceI cleavage to generate DSBs, SPOC1 & 53BP1 colocalize at distinct foci, including the cleaved DNA adjacent to DNA-bound GFP-LacR. Proteins were visualized by immunostaining & confocal microscopy. Scale bars = 10 μm. (B) Quantitative analysis of SPOC1 recruitment to the 53BP1 & GFP-LacR positive lacO array before & 16 h after I-SceI induction. (C) Monitoring of the kinetics of SPOC1 & 53BP1 recruitment to DSBs between 0 & 24 h post-I-SceI induction. (D) Quantifying ATM kinase inhibitor-mediated effects on recruitment of SPOC1 & 53BP1 to DSBs as evident 16 h after I-SceI-induced cleavage. (E) SPOC1 & 53BP1 colocalize at a large discrete endogenous repair focus as observed in some non-irradiated U2OS cells by immunostaining. Scale bar = 10µm. Image collected & cropped by CiteAb from the following publication (//academic.oup.com/nar/article-lookup/doi/10.1093/nar/gks868), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - A-type lamin depletion inhibits DNA repair & reduces cellular fitness to DNA damage. (A) Wild-type (WT) or Y259X HDF treated w/ proteasome inhibitor MG-132 (30 μm). Cell extracts prepared & levels of 53BP1 assessed by immunoblotting. beta -tubulin a loading control. WCE, whole cell extract. The relative levels of 53BP1 expressed relative to levels of untreated WT HDF (1.00). In untreated Y259X cells, 53BP1 levels fell to 15% & restored to ∽50% following treatment w/ MG-132. (B) Cell extracts prepared from WT & Y259X HDF & assessed by immunoblotting w/ indicated antibodies. Shown is a representative set of blots from two independent experiments. (C) WT & Y259X HDF biochemically extracted into cytoplasmic & nuclear fractions before immunoblotting for levels of 53BP1. (D) HDF & Y259X cells treated w/ IR (3 Gy), fixed at the times indicated & stained w/ gamma -H2AX antibody. The number of gamma -H2AX foci at each time point quantified & data represents the mean ± SD from 3 independent experiments. (E) U2OS cells transfected w/ siCTRL or siLMNA siRNA & plated onto 60-mm dishes. The cells then exposed to varying doses of IR & fixed 12 days later in a solution containing crystal violet. The surviving fraction at each IR dose calculated based on the plating efficiency of untreated plates. Data represent the mean ± SD from 3 independent experiments. (F) HDF or Y259X cells treated w/ DMSO or KU55933 ATM inhibitor (10 μm) before exposure to indicated doses of IR then fixed, processed & stained w/ gamma -H2AX antibody. Residual gamma -H2AX foci enumerated & the data represent the mean ± SD from 3 independent experiments. (G) HDF or Y259X cells exposed to IR, briefly extracted w/ CSK buffer & processed for IF w/ gamma -H2AX & pKAP-1 antibodies. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25645366), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - TRF2/Terc double null mutation induces DNA damage response resulting in terminal differentiation of metastatic tumorsHistopathology of primary SCC from K14Cre;TRF2+/+;Terc+/+ A. & K14Cre;TRF2f/f;Terc-/- C. mice is shown by H&E staining. Scale bar = 10 μm. Histopathology of metastatic SCC from K14Cre;TRF2+/+;Terc+/+ B. & K14Cre;TRF2f/f;Terc-/- D., E. mice is shown by H&E staining. Scale bar in (D) = 50 μm. Apoptotic cells in primary SCC from K14Cre;TRF2+/+;Terc+/+ F. & K14Cre;TRF2f/f;Terc-/- G. mice are shown by TUNEL assay. Scale bar = 10 μm. Proliferating cells were detected in K14Cre;TRF2+/+;Terc+/+ H. & K14Cre;TRF2f/f;Terc-/- I. primary SCC by PCNA immunohistochemistry & quantitated in J.. Error bars indicate SEM. Asterisk indicates statistically significant difference. Co-localization of 53BP1 (shown by immunofluorescence, AlexaFluor 488) at telomeres (shown by fluorescence in situ hybridization, Cy3) in histopathologic sections from K14Cre;TRF2+/+;Terc+/+ K. & K14Cre;TRF2f/f;Terc-/- L. primary SCC & quantitated in M. Error bars indicate SEM. Nuclei were counterstained with DAPI. Scale bar = 5 μm. N. Average telomere length ratios in CD34+ stem, Lgr6+ stem, & basal cells from K14Cre;TRF2+/+;Terc+/+ & K14Cre;TRF2f/f;Terc-/- primary SCC were determined by qPCR. Error bars represent SEM. O. Phospho-ATM, phospho-Chk2, & p53 expression in K14Cre;TRF2+/+;Terc+/+ & K14Cre;TRF2f/f;Terc-/- primary SCC is shown by western blot. beta -actin expression was used to control for equal loading of each lane. Representative blots are shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29113290), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Increased Neurogenesis in the Absence of INK4a/ARF Expression Is Independent of Apoptosis & Microglial Activation(A) Quantification of the number of apoptotic cells, as detected by TUNEL, in the DG 6 hr following exposure of WT, ink4a/arf-deficient (INK4a/ARF KO), & p53-deficient (p53 KO) mice to 6 Gy cranial IR. Indicated is the average number of apoptotic cells per DG section (n = 4 mice per group).(B) Quantification of CD68 expression in the DG 24 hr following exposure of mice to IR.(C) Representative confocal images showing CD68+ cells (in red) next to apoptotic TUNEL+ cells (in green) following exposure of WT mice to IR. Note the increase in CD68 signal & side by side localization of CD68+ & TUNEL+ cells following IR.(D–F) Proportion of neurons (NeuN, in green), young neurons (DCX, in green), & progenitor cells (Sox2, in green) showing persistent DNA damage foci (53BP1, in red) in the hippocampus of mice sacrificed 8 weeks post exposure to IR. Nuclei were stained with DAPI.(G) Representative confocal images of brain sections from irradiated WT mice displaying 53BP1 DNA damage foci with the indicated cell markers are shown. Images were acquired by a Leica microsystems TSC SP8 HyVolution confocal microscope & an HC PL APO CS2 40×/1.3 oil objective. The p values (∗p < 0.05; ∗∗∗p < 0.001) were obtained by performing a Student's t test. ND (no cells were detected). Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S2213671118301528), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - GamGFP marks DSBs in mammalian cells & is inhibited by Ku.(A) GamGFP co-localizes with 53BP1 on laser-induced DNA breaks. (B) Ku inhibits recruitment of GamGFP to laser-induced damage, live cells. (C & D) Ku inhibits recruitment of GamGFP, fixed cells. Mean ± SEM of three experiments, n >25 cells each. (E) GamGFP forms IR-induced foci in Ku80-defective MEFs. (F) Zoomed image from E. (G) IR-induced foci containing Gam only, 53BP1 only or both Gam & 53BP1 (>2600 total foci counted in three independent experiments). Error bars, SEM. Scale bars = 5 μm.DOI://dx.doi.org/10.7554/eLife.01222.012GamGFP marks DSBs in mammalian cells.(A) Live analysis of GamGFP localization to laser-induced DNA damage. Hela cells producing GamGFP were laser damaged along the cell track indicated by the red line at 0 min (m) & images were taken at the indicated times as shown. (B) GamGFP co-localizes with gamma H2AX in fixed Hela cells.DOI://dx.doi.org/10.7554/eLife.01222.013Ku inhibits GamGFP recruitment at DSBs independently of non-homologous end joining.Cells lacking either Ku80 or LigIV are defective in non-homologous end joining (NHEJ), yet the presence of Ku still inhibits recruitment of GamGFP to laser-induced DSBs even in NHEJ-defective cells, & thus independently of the cell's ability to complete NHEJ. Whereas it could have been possible that reduced GamGFP recruitment in the presence of Ku was caused by reduced persistence of DSBs due to their repair by NHEJ, our data show instead that Ku inhibits recruitment independently of successful NHEJ & imply that Ku binding to DSEs itself is inhibitory.DOI://dx.doi.org/10.7554/eLife.01222.014 Image collected & cropped by CiteAb from the following publication (//elifesciences.org/articles/01222), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Loss of functional PRPF31 induce RNA:DNA hybrid dependent genomic instability but not in mice retinal neurons.(a andb) gamma H2AX & 53BP1 foci analysis in PRPF31 siRNA-transfected RPE-1 cells. (c & d) gamma H2AX & 53BP1 foci analysis in vasculo-stromal fraction derived primary cells fromPrpf31+/A216P mice (Prpf31-ki). (e) gamma H2AX & 53BP1 foci analysis in retina fromPrpf31+/A216P mice on postnatal day 20. All column bars represent the mean. For (a-d) “n”, mentioned on respective column, signify number of cells analyzed from two independent experiments. For (e) n=16 for each column & signify number of retinal sections analyzed; acquired from n=4 eyes. Error bars represent Standard error of Mean (SEM). *P≤0.05; **P<0.01, ***P<0.001 using Mann-Whitney test (a,b), Kruskal-Wallis test followed by Dunn’s post hoc test (c,d); & two tailed unpaired Student’s t-test (e). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30345028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - Lamin A/C-53BP1 interaction is regulated in a DNA damage-dependent manner. (A) Left; HDF were subjected to IR (10 Gy) & allowed to recover for 1 h. Association between A-type lamins & 53BP1 was assessed by immunoprecipitation of endogenous lamin A/C followed by immunoblotting with 53BP1 & lamin A/C antibodies. WCE, whole cell extract, IP: immunoprecipitates. WCE represents 5% input. Right; endogenous lamin A/C was immunoprecipitated & supernatants or pellets were analysed by immunoblotting for interaction with JMJD2A. (B) U2OS/GFP-lamin A cells were pretreated with caffeine (20 mm) for 1 h before exposure to IR (10 Gy, 1 h recovery). Cell extracts were then subjected to immunoprecipitation using GFP-Trap beads, & bound complexes were then analysed by immunoblotting using 53BP1 & GFP antibodies. WCE represents 1% input. (C) As in (C) except cells were pretreated with 10 μm ATMi for 1 h before IR. WCE represents 1% input. (D) U2OS/GFP-lamin A cells were subjected to laser micro-irradiation, fixed 1 h later & immunostained with gamma -H2AX antibody. Scale bar, 10 μm. (E) U2OS cells were transfected with siCTRL or siLMNA & subjected to laser micro-irradiation, fixed 15 min later & then processed for immunofluorescence with gamma -H2AX & 53BP1 antibodies. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25645366), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Comparative kinetics of 53BP1 & SPOC1 recruitment to radiomimetic drug NCS-induced DSBs. U2OS cells fixed before (mock) or at the indicated times post-treatment (+NCS), immunostained for SPOC1 (green) & 53BP1 (red) & analyzed by confocal microcopy. At the earliest observation period after NCS treatment (15 min) a large number of small 53BP1 stained DSB repair foci emerged, but were not detectably stained by SPOC1. Between 2 to 24 h post-NCS treatment the number of small 53BP1 stained foci gradually, then almost completely disappeared, concomitant with emerging larger repair foci. At around 8 h post-NCS treatment most of the large 53BP1-stained repair foci were also strongly stained by SPOC1, suggesting SPOC1 is recruited to & accumulates at DSBs in heterochromatin known for their slow repair kinetics. Scale bars = 10µm. Image collected & cropped by CiteAb from the following publication (//academic.oup.com/nar/article-lookup/doi/10.1093/nar/gks868), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - RNA:DNA hybrid formation is central to ATM-53BP1 repair pathway.(a) Immunofluorescence using the anti-flag to quantify nuclear hybrid-binding (HB) domain foci signal in pre-permeabilized RPE cells & quantification of HB domain signal in cells treated with siATM, (b) Western blot showing ATM depletion. (c) Immunofluorescence & HB-domain foci quantification in cells treated with ATM inhibitor. (d) Quantitation of gamma H2AX foci in cells expressing HB domain or active RNAseH1, in presence or absence of H2O2 dependent oxidative stress. siC, non-targeted control siLuciferease RNA. Column bars represent the mean of n number of cells (described on each column) from three independent experiments. Error bars represent SEM. *P ≤ 0.05; **P < 0.01, ***P < 0.001 using Mann-Whitney test (a,c) & Kruskal-Wallis test followed by Dunn’s post hoc test (d). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30345028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - UMI acidic residues are required for RNF168-mediated site-specific ubiquitination & the DDR.a Sequence alignment analysis of RNF168 UMI domains across species. The evolutionary conserved E143/E144 are highlighted in red. b Deletion of residues 143-144 attenuates 53BP1 IRIF formation. U2OS RNF168 KO cells were transiently transfected with GFP-RNF168 or RNF168 delta 143-144. Cells were irradiated with 10 Gy, allowed to recover for 1 h, followed by immunofluorescence analysis with indicated antibodies. Repeated three times with similar results. c Deletion of 143-144 abolishes RNF168-mediated ubiquitination. Co-transfection of GFP-RNF168 or mutant with Flag-H2AX K13/K15 only vector in HEK293T cells followed by western blot analysis. Repeated three times with similar results. d RNF168 delta 143-144 is recruited to laser damage. U2OS RNF168 KO cells expressing GFP-RNF168 & RNF168 delta 143-144 were damaged using a 405 nm laser source & analyzed 15 min later by confocal microscopy. Dotted line indicates the laser path. Repeated three times independently with similar results. e–f U2OS cells were transfected with GFP-RNF168 wildtype & mutants. Proximity ligation assay (PLA) was performed using specific antibodies against GFP & gamma H2AX. Mean intensity of PLA signals in GFP-positive cells were measured & analyzed using ImageJ software. Data presented as mean ± SD. One-way ANOVA, Dunn’s multiple comparison test was used in statistical analysis, wildtype vs. R57D P = 0.1455, wildtype vs. delta 143-144 P = 0.1589, R57D vs. delta 143-144, P > 0.9999, ****P < 0.0001 with 95% confidence interval. n = 75 (wildtype), 48 (R57D), 74 ( delta 143-144), 53 (R57D/ delta 143-144) over 3 independent experiments. Source data are provided as Source Data file. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32424115), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - TRF2/Terc double null mutant mice exhibit DNA damage response at short telomeres in epidermisA. Average telomere length ratios in CD34+ stem, Lgr6+ stem, & basal cells from K14Cre;TRF2+/+;Terc+/+, K14Cre;TRF2f/f;Terc+/+, K14Cre;TRF2+/+;Terc-/-, & K14Cre;TRF2f/f;Terc-/- epidermis were determined by qPCR. Error bars represent SEM. Co-localization of 53BP1 (shown by immunofluorescence, AlexaFluor 488) at telomeres (shown by fluorescence in situ hybridization, Cy3) in histopathologic sections from K14Cre;TRF2+/+;Terc+/+ B., K14Cre;TRF2f/f;Terc-/- C., K14Cre;TRF2+/+;Terc-/- D., & K14Cre;TRF2f/f;Terc+/+ E. epidermis is shown. Nuclei are counterstained with DAPI. Scale bar = 5 μm. Phospho-ATM expression in histopathologic sections from K14Cre;TRF2+/+;Terc+/+ F., K14Cre;TRF2f/f;Terc-/- G., K14Cre;TRF2+/+;Terc-/- H., & K14Cre;TRF2f/f;Terc+/+ I. epidermis. Phospho-Chk2 expression in histopathologic sections from K14Cre;TRF2+/+;Terc+/+ J., K14Cre;TRF2f/f;Terc-/- K., K14Cre;TRF2+/+;Terc-/- L., & K14Cre;TRF2f/f;Terc+/+ M. epidermis. p53 protein expression in histopathologic sections from K14Cre;TRF2+/+;Terc+/+ N., K14Cre;TRF2f/f;Terc-/- O., K14Cre;TRF2+/+;Terc-/- P., & K14Cre;TRF2f/f;Terc+/+ Q. epidermis. Representative sections are shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29113290), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - Vismodegib increases radiation-induced gamma H2AX/53BP1 nuclear foci. BCC-1 or SCC-25 cells were treated for 24 h with 10 or 40 µM vismodegib or with DMSO as control followed by irradiation with 4 Gy. (A) Representative images of phosphorylated gamma histone-2AX ( gamma H2AX)/p53-binding protein 1 (53BP1) foci from non-irradiated & 4 Gy-irradiated DMSO control versus 40 µM vismodegib-treated cells are shown. Nuclei were counterstained with DAPI. Scale bar, 10 µm. (B) Quantification of persistent gamma H2AX/53BP1 foci in vismodegib treated & irradiated BCC-1 (left panel) & SCC-25 (right panel) cells. Bars represent means + SD from three independent experiments performed in duplicate. ** p < 0.01 vismodegib- versus DMSO-treated cells & #p < 0.05, ##p < 0.01 4 Gy versus non-irradiated cells (t-test). Gy, Gray; Vism., vismodegib. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30142876), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - The hypoxia-activated pro-drug Tirapazamine (TPZ) reduces stem-like Aldefluorbright population in vitro & in liver metastases(a) Human colonospheres cultured in hypoxia (0.1%) & normoxia (21%) for 24 hrs in absence or presence of TPZ for 4 hrs. Cells processed for IF analysis of gamma H2AX (green). DAPI (blue) used to visualize cell nuclei. (b) Bar graphs showing quantification of gamma H2AX foci observed in (a), shown as % of mean gamma H2AX intensity at normoxia (n=3). (c) Representative confocal pictures of comet assay of colonospheres cultured as in (a). (d) Bar graphs showing quantification of DNA damage observed w/ the comet assay (Extend tail moment) (n=77). (e) Experiment performed as in (a), but cells either treated or untreated for 16 hrs. Cells lysed & analyzed by WB for the indicated markers (cropped). (f) Colonospheres cultured in hypoxia & treated w/ TPZ for 16 hrs. The cells FACS sorted into Aldefluorbright & Aldefluordim populations & analyzed by WB for the indicated markers. (g) Human colonospheres cultured in hypoxia (0.1%) for 24 hrs in absence or presence of TPZ for 4 hrs & FACS sorted into Aldefluorbright & Aldefluordim populations. Bar graph the fold change Aldefluordim & Aldefluorbright cells upon TPZ treatment (n=3). (h) C26 tumor cells injected into liver parenchyma of immune-deficient mice. Following tumor initiation, tumor-bearing liver lobes subjected to a vascular clamping protocol [24] to induce hypoxia & treated w/ saline (control) or TPZ (treatment) for 10 days. At endpoint, livers excised & expression of ALDH1 examined by IHC. Magnification 20x, T=tumor. (i) Bar graphs showing quantification of ALDH1 (n=28) observed in (h) (n=14). * = significant (p>0.05). ns = not significant. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.21145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - MRE11 poly(T)11/12 sequence variant has no apparent functional consequence on DNA damage detection & repair.(A) Electropherograms & sequencing traces of the MRE11 poly(T)11 tract in leukemia/lymphoma cells: Daudi (heterozygous insertion of one bp, poly(T)11/12); Jurkat (heterozygous deletion of one bp, poly(T)10/11); & Raji (wild-type, poly(T)11). (B) MRE11 expression in Daudi, Jurkat & Raji cells. qRT-PCR was used to calculate the ratio of MRE11 expression (amplification of exons 2 to 4) relative to the GAPDH control, independently of the presence of the aberrant transcript observed in Jurkat. PCR products were loaded on the gel at saturation of the reaction, being not quantitative. MRE11 protein levels were assessed by Western blot, & were considerably lower in Jurkat. (C) ATM-pS1981 levels were assessed by Western blot in Daudi, Jurkat & Raji after 1.5 Gy irradiation of cells & 1 h, 4 h or 24 h recovery. There was an increase of ATM phosphorylation 1 h after irradiation in Daudi & Raji cells that decreased at the later time points. Jurkat showed no ATM phosphorylation after exposure to irradiation. (D) DNA repair foci was observed by immunofluorescence after 1.5 Gy IR followed by 1 h & 4 h recovery. Irradiation resulted in the formation of both MRE11 & 53BP1 foci in Daudi & Raji cells, whereas Jurkat failed to form MRE11 foci. Nuclei were counterstained with DAPI. Merged figure presents co-localization of MRE11 & 53BP1 foci visualized in yellow. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21637783), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Cry2−/− cells accumulate damaged DNA.(A) Representative early passage (P3–4) primary wildtype (WT), Cry1−/−, Cry2−/−, & Cry1−/−;Cry2−/− adult ear fibroblasts stained with anti-53BP1 antibody (green) & DAPI (blue). Insets show enlarged view of indicated cells. (B) Quantitation of 53BP1-positive cells prepared as described in (A). Nuclei containing more than five 53BP1 puncta & negative for BrdU labeling were considered positive for DNA damage. Data represent the mean ± s.d. for at least 200 cells per genotype. (C) Chi-squared analysis of the distributions of Cry1 & Cry2 wildtype (black), heterozygous (gray), & homozygous knockout (red) genotypes establishes a significantly reduced survival of Cry2−/− mice. **p < 0.01 by chi-squared analysis with 2 degrees of freedom (χ2 = 10.39).DOI://dx.doi.org/10.7554/eLife.04883.020 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25756610), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Persistence of repair foci at suboptimal pHe. (A) Left panel shows immunofluorescence detection of gamma H2AFX & 53BP1 following 24 h recovery from bleomycin treatment. Control, sample before damage induction. Colocalization is shown in white; PCC, Pearson correlation coefficient score. Right panel corresponding to gamma H2AFX foci/cell at 24 h & 48 h following recovery. Only foci larger than 10 pixels were counted. Experiments were performed in triplicate. Values are mean ± SEM. *P < 0.05, ****P < 0.00005. (B) Immunoblots for the detection of gamma H2AFX & histone H4. Right panel shows gamma H2AFX/H4 ratios at indicated pHe values. C, control before damage induction. Quantitation of three independent immunoblots is shown on the right. Values are mean ± SEM. *P < 0.05. (C) Immunofluorescence detection of gamma H2AFX after 48‐h incubation in range of pHe values tested without BLM treatment. Right panel corresponding to gamma H2AFX foci/cell at 48 h of pHe incubation. Only foci larger than 10 pixels were counted. Experiments were performed in triplicate. Values are mean ± SEM. *P < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29511618), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - BETd blocks 53BP1 function in senescent cells.a Early passage (control) or late passage (replicative senescence) TIG-3 cells were treated with 10 nM ARV825 (+) or vehicle (−) for 4 days. These cells were then subjected to western blotting using the antibodies shown on the right after immunoprecipitation with the antibodies shown at the top of the panel (IP:). b–f Early passage (control) or late passage (replicative senescence) TIG-3 cells were transfected with previously validated two different siRNA oligos against 53BP1 or control siRNA oligo twice at 2 days intervals. These cells were then subjected to RT-qPCR analysis (b), western blotting analysis (c), the cell proliferation analysis (d), immunofluorescence staining using the antibodies shown on the left (e) or neutral comet assay (f). Representative photographs of the cells in the indicated culture conditions are shown & the histogram shown at the bottom of the panel indicates the relative cell number (d). The number of gamma H2AX foci, above threshold intensity per nucleus (n = 50) was quantified & shown at the bottom of the e. The average tail olive moments were shown at the bottom of the f. Each box indicates the median & 25% & 75% quantiles, & the whiskers represent the minimum & maximum observations (e, f). For all graphs, error bars indicate mean ± s.d. (n = 3 for b, n = 4 for d) & the representative data from three independent experiments were shown. Statistical significance was determined with two-tailed unpaired Student’s t-test (b), one-way ANOVA followed by Tukey multiple comparison test (d), one-way ANOVA followed by Holm-Sidak multiple comparison test (e, f). P values < 0.05 were considered significant. Source data are provided as a Source Data file. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32321921), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Bystander signaling evokes DNA damage foci formation in urothelial cancer & normal urothelial cells(A) Schematic representation of experimental conditions including non-irradiated, uniformly-irradiated & shielding protocols where 50% of the slide was exposed & 50% shielded with MCP alloy. Cells were fixed & processed for 53BP1-immunofluorescence, one hour following irradiation. Micrographs of urothelial cancer T24 cells labelled with anti-53BP1 (green) to label nuclear DNA double stand break foci & DAPI to counterstain nuclei are shown for each experimental condition. (B) Summary graph of mean foci/cell in T24 cells (N=3), measured from confocal z-stacks in Volocity software. There were significantly more foci in shielded cells 0.5mm from the shield than in non-irradiated controls, indicative of a bystander effect (p=0.019). (C) Summary data for HT1376 cancer cells (N=3) where there was no difference in the mean foci/cell between non-irradiated controls & shielded cells. (D) Summary data for normal urothelial HUC (N=3). Mean foci/cell was significantly greater in shielded cells in the 0-5mm (p=0.005) & 5-10mm (p=0.04) analysis regions from the shield. * denotes p<0.05, ** denotes p<0.01 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29228614), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Pot1a prevents DDR in HSCs. a, b Telomeric DDR in 8 week-old LSK cells upon Pot1a knockdown. a Immunocytochemical staining of TRF1 (green) & 53BP1 (red). Foci co-stained with TRF1 & 53BP1 were identified as TIFs. Nuclei were stained with TOTO3 (blue). Scale bar, 2 μm. b Frequency of TIFs per cell after 1 week of culture. Data are expressed as the mean ± SD (n = 100, *p < 0.01 by t-test). Representative data from three independent experiments are shown. c–f Telomeric DDR in donor-derived control-GFP or Pot1a overexpressing 8 week-old LSK cells 4 months post BMT. c Immunocytochemical staining of TRF1 (green) & 53BP1 (red). Nuclei were stained with TOTO3 (blue). Scale bar, 2 μm. d Frequency of TIFs per cell. Data are expressed as the mean ± SD (n = 120, *p < 0.01 by t-test). Representative data from 3 independent experiments are shown. e Immunocytochemical staining of TRF1 (green) & RPA32 (red). f Immunocytochemical staining of TRF1 (green) & pChk1 (red). Scale bar, 2 μm. g Flow cytometric analysis of Chk1 & pChk1 in donor-derived GFP+ LSKCD48–CD150+ cells after 5 months of 2nd BMT. Mean fluorescence intensity of Chk1 & pChk1 (left panels). Data are expressed as the mean ± SD (n = 3, *p < 0.01 by t-test). Representative FACS profiles of Chk1 & pChk1 in donor-derived GFP+ LSKCD48–CD150+ cells (right panels) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28986560), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - Aphidicolin-induced ATM signaling in immortalized MEFs. (A) Cells (WT, MJI-53; KO, MJI-1) were treated for 24 h using 0 or 3 μM aphidicolin. (B) Cells (lane 1–3, WT, MJI-91; lanes 4–6, KO, MJI-186; lanes 7–9, WT, MJI-105; lanes 1–12, KO, MJI-84) were treated for 24 h using 0, 1 or 3 μM aphidicolin. Actin & 53BP1 serve as loading controls. DYNLL1 is used as a surrogate marker for loss of ASCIZ. (C) Quantification of western blot band intensities. An arbitrary unit of 100 represents the average band intensity for the respective phospho-protein in the 3 μM aphidicolin-treated wildtype samples on each membrane. Graphs indicate the mean ± standard error, n = 3. Additional loading controls for total KAP1, p53 & H2AX are shown in Supplementary Fig. S1A & B. Image collected & cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S1568786417301349), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - POT1 prevents DDR in human HSC. a Expression of POT1 in hCB CD34+ cells & LT-HSCs (Lin–CD34+CD38−CD45RA−CD90+CD49f+). b hCB-derived LT-HSCs were cultured with MTM-POT1. After 10 days of culture, cells were isolated & re-cultured in methylcellulose medium (200 cells per dish). The number of CFU-C & HPP-CFC (>1.0 mm, >2.0 mm) are shown. Data are expressed as the mean ± SD (n = 3, *p < 0.01 by t-test). Representative data from three independent experiments are shown. c, d hCB LT-HSCs were cultured for 10 days with control MTM protein or MTM-POT1. After 10 days of culture, LT-HSCs were re-isolated & number of TIF was examined. c Immunocytochemical staining of TRF1 (green), 53BP1 (red), & TOTO3 (blue). Scale bar, 2 μm (left). Frequencies of TIFs after 10 days of culture (right). Data are expressed as the mean ± SD (n = 100: control, n = 100: MTM-POT1, *p < 0.01 by t-test). Representative data from 2 independent experiments are shown. d Immunocytochemical staining of TRF1 (green), RPA32 (red), & TOTO3 (blue). Scale bar, 2 μm (left). Frequencies of TIFs after 10 days of culture (right). Data are expressed as the mean ± SD (n = 110–120: control, n = 110: MTM-POT1, *p < 0.01 by t-test). Representative data from two independent experiments are shown Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28986560), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Telomere-initiated senescent cells retain active DDR foci for years after senescence establishment.a. DDR, in the form of ATM pS1981 foci co-localizing with 53BP1 & gamma H2AX foci, is detectable three years after senescence establishment. Scale bar, 10 µm. Below, bar graphs show the percentage of cells positive ± s.e.m. for the indicated DDR markers, in senescent (sen), early passage proliferating (prol) or telomerized proliferating (tel) skin fibroblasts from two independent centenarian donors (cen2 & cen3). Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). b. SA-beta -gal staining of the two batches, cen2 & cen3, is shown together with the percentage of BrdU-positive cells. Scale bar, 100 µm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0110969), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - VPA at a safe dose can lead to the dysfunction of DNA repair function. (A) The images represent gamma H2AX & 53BP1 foci formation in U2OS cells treated with 0.5 or 1.0 mM VPA at 24 h post-IR treatment (left panel), & the percentage of cells with gamma H2AX foci or 53BP1 foci formation in each group was calculated (right panel, the cell with >10 foci was called positive & counted). DAPI was used for nuclear staining; (B) The relative DNA damage in primary-culture tumor cells at 120 min post-IR was analyzed by comet assay (upper), & gamma H2AX foci formation in primary-culture tumor cells at 24 h post-IR was presented，“+” & “−“ indicated whether VPA was added in the groups (lower left) & calculated (lower right); (C) The clonogenic survival assay was used to detect survival in the U2OS cells treated with the combination of 0.5 or 1.0 mM VPA with 10 μM ABT888 (PARPi). Each data point in the graphs was from three independent experiments (mean ± SD). p-Values were calculated by Student’s t-test (* p < 0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28489060), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - The susceptibility of human LECs to low-dose ionizing radiation. The human lens cell line FHL124 was exposed to low-dose IR up to 2.28 Gy. Exposed cells were then processed for both immunofluoresence microscopy (a) & immunoblotting (b) 1 h later. The signals obtained by immunoblotting were quantified & the mean from three independent experiments calculated & plotted (c) against IR dose. GAPDH was used as a loading control. Both gamma H2AX & RAD51 increased linearly with IR dose. Signals for other markers of DNA repair, 53BP1, MRE11 & TP53, remained unchanged as assessed by immunoblotting (b). By immunofluoresence microscopy (a), MRE11 & 53BP1 redistributed into nuclear foci, particularly at the 2.28 Gy level. TP53 remained uniformly distributed throughout the nuclear compartment, but excluded from nucleoli (a). As the levels of gamma H2AX & RAD51 increased after exposure to IR (c), so the number of nuclear foci also increased (a). Scale bar, 10 µm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25924630), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - A-type lamin depletion inhibits DNA repair & reduces cellular fitness to DNA damage. (A) Wild-type (WT) or Y259X HDF treated w/ proteasome inhibitor MG-132 (30 μm). Cell extracts prepared & levels of 53BP1 assessed by immunoblotting. beta -tubulin a loading control. WCE, whole cell extract. The relative levels of 53BP1 expressed relative to levels of untreated WT HDF (1.00). In untreated Y259X cells, 53BP1 levels fell to 15% & restored to ∽50% following treatment w/ MG-132. (B) Cell extracts prepared from WT & Y259X HDF & assessed by immunoblotting w/ indicated antibodies. Shown is a representative set of blots from two independent experiments. (C) WT & Y259X HDF biochemically extracted into cytoplasmic & nuclear fractions before immunoblotting for levels of 53BP1. (D) HDF & Y259X cells treated w/ IR (3 Gy), fixed at the times indicated & stained w/ gamma -H2AX antibody. The number of gamma -H2AX foci at each time point quantified & data represents the mean ± SD from 3 independent experiments. (E) U2OS cells transfected w/ siCTRL or siLMNA siRNA & plated onto 60-mm dishes. The cells then exposed to varying doses of IR & fixed 12 days later in a solution containing crystal violet. The surviving fraction at each IR dose calculated based on the plating efficiency of untreated plates. Data represent the mean ± SD from 3 independent experiments. (F) HDF or Y259X cells treated w/ DMSO or KU55933 ATM inhibitor (10 μm) before exposure to indicated doses of IR then fixed, processed & stained w/ gamma -H2AX antibody. Residual gamma -H2AX foci enumerated & the data represent the mean ± SD from 3 independent experiments. (G) HDF or Y259X cells exposed to IR, briefly extracted w/ CSK buffer & processed for IF w/ gamma -H2AX & pKAP-1 antibodies. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25645366), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - UDM1 is required for RNF168-mediated 53BP1 recruitment.a Schematic illustration of RNF8 & RNF168 structural domain organization, RNF168 fragments, & chimeric proteins used as in b & c. FHA–forkhead-association domain; coiled-coil–coiled-coil domain; RING–ubiquitin E3 ligase RING domain; Rs-arginine anchor; LRM–LR motif; UMI–UIM-and MIU-related ubiquitin binding domain; MIU–motif interacting with ubiquitin. b–c Transient transfection of GFP-tagged RNF168 fragments & chimeric proteins in U2OS RNF168 KO cells. Cells were irradiated with 10 Gy & allowed to recover for 1 h followed by immunofluorescence analysis using 53BP1 antibody as indicated. Cells were counterstained with DAPI. Repeated at least two times independently with similar results. Source data are provided as Source Data file. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32424115), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: 53BP1 Antibody - BSA Free [NB100-304] - Loss of ATM function in retina.(a) Retinal explants pretreated with ATM inhibitor (ATMi) or PI3K inhibitor (DDRi) are irradiated & sections analyzed for gamma H2AX. (b) ATM knockout adult mice retina analyzed for irradiation dependent gamma H2AXaccumulation. (c) Western blot analysis of ATM & 53BP1 in micro-dissected neuronal retina. LC, Coomassie-equivalent staining of gels used as loading control (representation of n=2 independent repeats). (d) Cartoon depiction of domains of ATM protein. (e) Neural retina of mice was microdissected & cDNA was prepared. PCR performed using indicated domain-specific primers onATM mRNA. Unlike FATC & TAN domain coding mRNA; the FAT-domain containing mRNA could be observed even in 5-month-old adult mice retina, indicating the presence of an alternatively spliced form of ATM in the retina. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30345028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - TIFs measured in leukocytes. (A) The percentage of cells with one or more TIFs in griffon vultures. The age & number of cells measured for each individual was as follows: 8.06 years: 77 cells, 10 years: 29 cells, 10.9 years: 153 cells, 12.3 years: 146 cells, 15.3 years: 125 cells, & 21.4 years: 173 cells. (B) The percentage of cells with one or more TIFs in American flamingos. The age & number of cells measured for each individual was as follows: 0.79 years: 3 cells, 1.75 years: 12 cells, 1.8 years: 34 cells, 6.75 years: 21 cells, 13.8 years: 11 cells, 16.9 years: 9 cells, 19.9 years: 7 cells, 19.9 years: 7 cells, 34.9 years: 20 cells, & 35.9 years: 6 cells. In the representative images, the nuclei are stained blue with DAPI, the telomeres are red, & the 53BP1 stain is in green. 53BP1 staining at the very edge of nuclei was not counted as foci. The top row of the representative images shows maximum projections which are the result of taking the maximum value of several different z-planes. The magnification image is displayed for a single plane rather than a maximum projection to show co-localization of the stains. A white arrow indicates a colocalization of 53BP1 & a telomere spot (a TIF). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31730540), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - NRK1 deletion curbs PARP activity & exacerbates DNA damage. a NAD+ levels in the liver of NRK1 LKO mice upon LFD & HFD (n = 8 for all groups except NRK1 LKO on HFD, n = 10; 24 weeks of age). b NR (left), NMN (center) & NAM (right) levels in the liver of NRK1 LKO & control mice upon HFD (n = 6 mice per group). c mRNA level of genes involved in the NAD+ biosynthesis pathways (n = 8 mice for the control group, n = 10 mice for the NRK1 LKO group). d Levels of poly(ADP-ribose) protein modification, PARP1, SIRT1, NAMPT, & GAPDH in the liver of NRK1 LKO & control mice on HFD. e Representative staining (top) & quantification (bottom) of gamma H2AX (left) & 53BP1 (right) immunofluorescence on liver sections from NRK1 LKO & control mice on HFD (n = 4 mice per group, ×10 magnification, DAPI counterstaining, scale bars: 200 μm). Results shown are mean ± SEM, * & *** indicate statistical difference vs. control group at p < 0.05 & p < 0.001, respectively. The individual values & statistical tests used for each panel can be found in the Source Data file Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31541116), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - DNA repair response to irradiation-induced DNA-breaks in adult mice retina.(a) Immunofluorescence performed using anti-gamma H2AX & 53BP1 antibodies in mice retina 1hr after exposure to 5 Gyrase of ionizing radiation. gamma H2AX appears in all cell types in response to DNA breaks. 53BP only observed in the ganglion cell layer (GC) & inner strata of inner nuclear layer (INL) (seeFigure 1a). Zoom in show foci formation in cells expressing 53BP1. (b) H2AX phosphorylation in all retinal cell types is euchromatin specific. (c) Kinetics of DNA repair in mice retina. Post-Irradiation, mice were sacrificed at indicated times & retinal sections were analysed for gamma H2AX & 53BP1. Left panel show all nuclear layers of retina;right panel show zoom in images to emphasize foci formation. Two mice were used for each condition, which were processed & stained together. Random image were taken using confocal microscope on each eye. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30345028), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Detection of 53BP1 in CTCs from MBC patients in the DETECT IV trial. (a) Selection of MBC patients with CTC-positivity (HER2-) & primary TNBC or primary HER2-, HR+ breast cancer for Eribulin monotherapy & blood sample collection. Blood samples were drawn before therapy (baseline visit), four, twelve, & 24 weeks after the start of Eribulin treatment (1st, 2nd, & 3rd treatment visit) as well as one year after treatment initiation or during premature termination due to disease progression (final visit). The numbers of patients recruited at the different visits are indicated by black columns, the numbers of CTC+ & CTC- patients among them by red & grey columns, respectively. Note that not all of the patients described in Table 1 entered the study during the baseline visit but during later visits which explains the differences in patient numbers. (b) Evaluation of 53BP1 signals in CTCs. Following EpCAM-based CTC enrichment & immunofluorescence microscopic imaging (Cellsearch®), CTCs (CK+, CD45-) were enumerated & 53BP1-staining intensity assessed per individual CTC as indicated by representative examples. A 53BP1 score per patient sample was calculated from the percentage of CTCs without & with light, moderate or strong staining. PBMC, peripheral blood mononuclear cell (CK-, CD45+). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32283863), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Treatment w/ exogenous POT1 protein protects LT-HSCs in culture. a Effect of MTM-POT1a on colony formation of HSCs. LT-HSCs (8 week-old) cultured w/ MTM-POT1a for 2 weeks. After culture, Lin− cells isolated & re-cultured in MethoCult™ GF M3434 medium (200 cells per dish). Data are expressed as the mean ± SD (n = 3, *p < 0.01 by Tukey’s test). Representative data from two independent experiments are shown. b, c LT-HSCs (8 week-old) cultured w/ MTM-POT1a or control MTM protein. b Number of total cells & LT-HSCs on day 4, 7, & 10 of culture. Data are expressed as the mean ± SD (n = 6, *p < 0.01 by t-test). c Percentage of Annexin V+PI+ apoptotic cells in LT-HSCs on day 4, 7, & 10 of culture. Data are expressed as the mean ± SD (n = 6, *p < 0.01 by t-test). d LT-HSCs (8 week-old) transduced w/ control shRNA or shPot1a-1. After 2 days of shRNA transduction, GFP+LT-HSCs re-sorted & cultured w/ MTM-POT1a or control MTM protein. After 1 week of culture, Annexin V assay performed. Percentage of Annexin V+PI+ apoptotic cells in GFP+LT-HSC fraction is shown. (n = 3, **p < 0.05 by Tukey’s test). e Immunocytochemical staining of TRF (green), 53BP1 (red), & TOTO3 (blue) in 8 week-old LT-HSCs cultured for 3 weeks (left). Frequencies of TIFs after 3 weeks of culture (right). Data are expressed as the mean ± SD (n = 80–100, *p < 0.01 by t-test). f Representative FACS profiles of 8 week-old LT-HSCs after 3 weeks of culture w/ control MTM protein or MTM-POT1a (left). Numbers of LSK cells & LT-HSCs after 3 week culture (starting from 2400 cells) are shown in right panels. Data are expressed as the mean ± SD (n = 3, *p < 0.01 by t-test). Representative data from five independent experiments are shown Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28986560), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Induction of senescence in hepatocytes causes mitochondrial dysfunction & reduces fatty acid oxidation capacity.Hepatocytes isolated from wild-type C57Bl/6 mice acquire a senescent phenotype 6 days after 10 Gy X-ray irradiation: (a) representative images showing SA-beta -Gal in non-irradiated cells (NOIR) & 6 days after irradiation, Merge: light blue=DAPI, dark blue=SA-beta -Gal, scale bar, 20 μm; (b) frequencies of SA-beta -Gal-positive hepatocytes & (c) representative images showing 53BP1 staining in NOIR & 6 days after irradiation. Merge: blue=DAPI, red=53BP1, scale bar 10 μm; (d) average number of 53BP1 foci per cell are significantly increased 6 days after irradiation. (e) Representative images showing Nile red staining in NOIR & 6 days after IR. Merge: blue=DAPI, red=Nile red, scale bar scale bar 20 μm. (f) Average fluorescence intensity of Nile red staining in senescent or non-senescent hepatocytes. (g) Change in OCR in senescent or non-senescent hepatocytes after addition of the fatty acid palmitate as substrate; (h) change in OCR in senescent or non-senescent hepatocytes after inhibition of fatty acid oxidation by etomoxir. Data in b,g,h are mean±s.e.m. & in d,f are mean±s.d. with 3–4 animals per group. Significant differences (t-test) are indicated with *P≤0.05 & **P≤0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28608850), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

An intracellular stain was performed on U-2 OS human osteosarcoma cell line with Rabbit anti-53BP1 Affinity Purified Polyclonal Antibody conjugated to Alexa Fluor ® 647 (Catalog # NB100-304AF647, blue histogram) or matched control antibody (Catalog # NBP2-24981AF647, orange histogram) at 2.5 µg/mL for 30 minutes at RT.

53BP1 was detected in immersion fixed SJCRH30 human Rhabdomyosarcoma cell line using Rabbit anti-53BP1 Antigen Affinity Purified Polyclonal Antibody conjugated to Alexa Fluor® 488 (Catalog # NB100-304AF488) (green) at 5 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.

Reactivity:

Hu, Mu, Rt, Po, Pm, Fe, Pm-Or, Rb, Sh

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Pm, Fe, Pm-Or, Rb, Sh

Applications:

, , , , , ,

Applications: , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-304AF350
Alexa Fluor 405	NB100-304AF405
Alexa Fluor 488	NB100-304AF488
Alexa Fluor 532	NB100-304AF532
Alexa Fluor 594	NB100-304AF594
Alexa Fluor 647	NB100-304AF647
Alexa Fluor 700	NB100-304AF700
Alexa Fluor 750	NB100-304AF750
Alexa Fluor Plus 405	NB100-304AFP405
Alexa Fluor Plus 488	NB100-304AFP488
Alexa Fluor Plus 555	NB100-304AFP555
Alexa Fluor Plus 594	NB100-304AFP594
Alexa Fluor Plus 647	NB100-304AFP647
Alexa Fluor Plus 680	NB100-304AFP680
Allophycocyanin	NB100-304APC
Allophycocyanin/Cy7	NB100-304APCCY7
Biotin	NB100-304B
CoraFluor 1	NB100-304CL1
DyLight 350	NB100-304UV
DyLight 405	NB100-304V
DyLight 488	NB100-304G
DyLight 550	NB100-304R
DyLight 594	NB100-304DL594
DyLight 650	NB100-304C
DyLight 680	NB100-304FR
DyLight 755	NB100-304IR
FITC	NB100-304F
HRP	NB100-304H
Janelia Fluor 525	NB100-304JF525
Janelia Fluor 549	NB100-304JF549
Janelia Fluor 646	NB100-304JF646
Janelia Fluor 669	NB100-304JF669
PE	NB100-304PE
PE/Atto594	NB100-304PEATT594
PE/Cy5.5	NB100-304PECY55
PE/Cy7	NB100-304PECY7
PerCP	NB100-304PCP
mFluor Violet 450 SE	NB100-304MFV450
mFluor Violet 500 SE	NB100-304MFV500
mFluor Violet 610 SE	NB100-304MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-304SS

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NB600-501

beta-Actin Antibody (AC-15)

(27 Reviews)

(644 Publications)

0.1 ml

NB600-501

<b>Left:</b> Simple Western lane view shows lysates of mouse brain, loaded at 1 mg/ml. A specific band was detected for beta ‑Actin at approximately 47 kDa (as indicated) using both 1:500 and 1:100 of Mouse Anti-Human/Mouse/Rat beta ‑Actin Monoclonal Antibody (Catalog # NB600-501) followed by HRP-conjugated Goat Anti-Mouse Secondary Antibody (Catalog # 042-205). This experiment was conducted under reducing conditions and using the 12-230kDa separation system. <b>Right:</b> Simple Western electropherogram showing the same Mouse Anti-Human/Mouse/Rat beta ‑Actin Monoclonal Antibody (Catalog # NB600-501) tested at 1:500 (blue line) and 1:100 (green line) in the mouse brain.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Beta-Actin expression in Fibroblast Hs68 cell lysate using anti-beta-Actin antibody. Image from verified customer review.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Cell line lysates were separated on SDS-PAGE and probed with 1:5000 Monoclonal Anti-beta-Actin Clone: AC-15 . The antibody was developed using Goat Anti-Mouse IgG-Peroxidase and a chemiluminescent substrate.Lanes: 1. HeLa, 2. JURKAT, 3. COS7, 4. NIH-3T3, 5. PC-12, 6. RAT2, 7.CHO, 8. MDBK, 9. MDCK

Immunocytochemistry/Immunofluorescence: beta-Actin Antibody (AC-15) [NB600-501] - Analysis of beta-Actin antibody on mouse fibroblasts. Image from verified customer review.

Immunohistochemistry: beta-Actin Antibody (AC-15) [NB600-501] - Staining of formalin-fixed, paraffin-embedded human heart sections with 1:1000 Monoclonal Anti-beta-Actin Clone: AC-15.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Analysis of beta-Actin in bovine whole skeletal muscle lysate. Image from verified customer review.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Whole cell extract of human fibroblasts was separated on SDS-PAGE and blotted with Monoclonal Anti-beta-Actin. The antibody was developed with Goat Anti-Mouse IgG, Peroxidase conjugate and AEC substrate. Lanes A: Antibody dilution 1:5,000 B: Negative control (only secondary antibody).

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - beta-Actin expression in human glioma cells using anti-beta-Actin antibody. Image from verified customer review.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - HEK293T cells transfected with Niaph virus Matrix Protein, expression 24h later. This image was submitted via customer review.

<b>Genetic Strategies Validation. </b>beta-Actin-Antibody-AC-15-Western-Blot-NB600-501-img0034.jpg

beta-Actin-Antibody-AC-15-Western-Blot-NB600-501-img0035.jpg

beta-Actin-Antibody-AC-15-Western-Blot-NB600-501-img0037.jpg

Immunocytochemistry/Immunofluorescence: beta-Actin Antibody (AC-15) [NB600-501] - Cultured FS-11 human fibroblast cell line stained using Monoclonal Anti-Beta-Actin, Clone No. AC-15 (NB600-501).

Immunocytochemistry/Immunofluorescence: beta-Actin Antibody (AC-15) [NB600-501] - HS-68 cells.

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - Analysis using the HRP conjugate of NB600-501. Electropherogram image(s) of corresponding Simple Western lane view. Beta-Actin antibody was used at 1:500 dilution on Hela, MCF-7, SH-SY5Y, & Jurkat lysate(s).

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - Analysis using the HRP conjugate of NB600-501. Image shows a specific band for Beta-Actin in 0.2 mg/mL of Hela, MCF-7, SH-SY5Y, & Jurkat lysate(s). This experiment was performed under reducing conditions using the 12-230 kDa separation system. * Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody.

VPA reduces autophagy at P28. (A) Schematic of VPA administration. (B) Representative Western blot images for LC3B, p62, p-rpS6 S240/244, DARPP32, & actin. (B–G) Quantification of (B–D) p-rpS6 S240/244 relative to actin, (B,E,F) LC3B-ii relative to actin (B,G,H) p62 & (B,I,J) DARPP32 normalized to VPA untreated (veh, vehicle) mice at P10 & P28, respectively. Data analyzed with unpaired, two-tailed t-test; (C) p-rpS6 S240/244/actin at P10, ns; (D) p-rpS6 S240/244/actin at P28, t11 = 2.882, p = 0.0149 (E) LC3B-ii/actin at P10, ns; (F) LC3B-ii/actin at P28, t10 = 2.231, p = 0.047; (G) p62 at P10, ns; (H) p62 at P28, t10 = 3.752, p = 0.0038; (I) DARPP32 at P10, ns, & (J) DARPP32 at P10, ns, *p < 0.05, **p < 0.01, ns not significant, n = 4–7 mice/treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32296308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Hypoxia causes DNA damage in patient-derived colonosphere cultures in vitro(a) Two independent patient-derived colonospheres were cultured in normoxia (21%) or hypoxia (0.1%) for 24 hours. Immunofluorescence was then used to asses gamma H2AX (green) in the nuclei DAPI (blue). (b) Bar graphs showing the quantification of gamma H2AX observed in (a) (n=3), shown as percentage of the mean gamma H2AX intensity at normoxia. (c) Representative confocal pictures of the comet assay of colonospheres cultured as in (a). (d) Bar graphs showing the quantification of DNA damage observed with the comet assay (Extend tail moment) (n=73). (e) Colonospheres were cultured as in (a) & gamma H2AX was assessed by FACS. The plots show normoxic gamma H2AX levels in red & hypoxic gamma H2AX levels in blue in two independent cell lines. (f) Human colonospheres were exposed to hypoxia for the indicated periods of time. Cell lysates were then analyzed by Western blotting for the indicated markers (cropped). Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.21145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Vps34 activity is required for maintaining LC3B-ii levels at P10 & mTOR inhibition increases p-Beclin-1 & LC3B-ii levels at P28. (A) Representative Western blot images for actin, DARPP32 & LC3B-i & -ii in striatal slices obtained from P10 or P28 mice, treated with SAR405 (1 μM) or vehicle (Veh; DMSO, 0.1%). (B,C) Quantification of LC3B-ii relative to actin, normalized to vehicle condition at each age. (B) P10: unpaired, two-tailed t-test, t11 = 2.985, p = 0.0124; P28: unpaired, two-tailed t-test, t24 = 1.807, p = 0.0922. P10: Veh: n = 6 slices, SAR405 n = 7 slices from 3 to 4 mice/age. (C) P28: Veh: n = 9 slices, Baf n = 7 slices from 3 to 4 mice/age. *p < 0.05. (D) Representative Western blot images for actin, DARPP32, p-Beclin-1 Ser14 & LC3B-i & -ii in striatal slices from P28 mice, treated with Torin-1 (5 μM) or vehicle (Veh; DMSO, 0.1%). Quantification of (E) LC3B-ii & (F) p-Beclin S14 relative to actin, normalized to vehicle. Data analyzed with unpaired, two-tailed t-test; (E) LC3B-ii/actin, t24 = 2.853, p = 0.0088. Veh: n = 19 slices, Baf n = 7 slices from 5 to 7 mice. *p < 0.05. (F) p-Beclin S14/actin, t22 = 3.337, p = 0.0030. **p < 0.01, Veh: n = 18 slices, Torin-1 n = 6 slices from 3 to 5 mice. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32296308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

The hypoxia-activated pro-drug Tirapazamine (TPZ) reduces stem-like Aldefluorbright population in vitro & in liver metastases(a) Human colonospheres cultured in hypoxia (0.1%) & normoxia (21%) for 24 hrs in absence or presence of TPZ for 4 hrs. Cells processed for IF analysis of gamma H2AX (green). DAPI (blue) used to visualize cell nuclei. (b) Bar graphs showing quantification of gamma H2AX foci observed in (a), shown as % of mean gamma H2AX intensity at normoxia (n=3). (c) Representative confocal pictures of comet assay of colonospheres cultured as in (a). (d) Bar graphs showing quantification of DNA damage observed with the comet assay (Extend tail moment) (n=77). (e) Experiment was performed as in (a), but cells were either treated or untreated for 16 hrs. Cells were lysed & analyzed by WB for the indicated markers (cropped). (f) Colonospheres cultured in hypoxia & treated with TPZ for 16 hrs. The cells were FACS sorted into Aldefluorbright & Aldefluordim populations & analyzed by WB for the indicated markers. (g) Human colonospheres cultured in hypoxia (0.1%) for 24 hrs in absence or presence of TPZ for 4 hrs & FACS sorted into Aldefluorbright & Aldefluordim populations. Bar graph shows the fold change Aldefluordim & Aldefluorbright cells upon TPZ treatment (n=3). (h) C26 tumor cells were injected into the liver parenchyma of immune-deficient mice. Following tumor initiation, tumor-bearing liver lobes were subjected to a vascular clamping protocol [24] to induce hypoxia & treated with saline (control) or TPZ (treatment) for 10 days. At endpoint, livers were excised & expression of ALDH1 was examined by IHC. Magnification 20x, T=tumor. (i) Bar graphs showing quantification of ALDH1 (n=28) observed in (h) (n=14). * = significant (p>0.05). ns = not significant. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.21145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effect of IFN-gamma pretreatment on carfilzomib sensitivity of H727 (a) & H23 (b) cells. IFN-gamma (150 U⋅ml−1) pretreatment for 24 h led to increased expression (left panel) & activity (middle panel) of proteasome immuno-subunits, & sensitized H727 cells to Cfz, while desensitizing H23 cells towards Cfz. The IC50 values displayed a statistically significant difference between IFN-gamma -pretreated cells & vehicle control (P value < 0.01, n = 3, Student’s t-test comparing the log transformed IC50 values obtained from three independent runs). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30858500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - RARA overexpression increases chemosensitivity to AM80(A, B) Overexpression of either RARAwt or RARAR394Q increases chemosensitivity of (A) HuT78 & (B) Karpas 299 cells to AM80. The presence of the R394Q mutation does not reduce chemosensitivity significantly. (C) RARAwt overexpression increases retinoic acid response element (RARE) activity (indicated by luciferase) in HuT78 cells treated with AM80. (D) RARAwt overexpression increases the degree of CDK2, CDK4 & CDK6 inhibition caused by AM80. wt, wild-type; CDK, cyclin-dependent kinase. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.15441), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - RARA drives expression of cyclin-dependent kinases(A) Knockdown of RARA causes G1 cell cycle arrest (P = 0.004) in Mac-1 cells. (B) Expression of the regulators of cell cycle progression, CDK6, CDK4, & to a lesser extent, CDK2, is inhibited by RARA knockdown in Mac-1 cells. (C) Expression of CDK6, CDK4, & CDK2 is increased following overexpression of RARAwt & RARAR394Q in HuT78 cells. RARA, retinoic acid receptor alpha; wt, wild-type; siRNA, small interfering RNA; CDK, cyclin-dependent kinase. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.15441), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - (A) Protein expression level of CCL2 with WARS silencing under control & IFN gamma stimulation conditions, performed on human primary PBMC-derived macrophages. *p<0.05, Student’s t-test(B) Western blot analysis showing the protein levels of phosphorylation of STAT1 with WARS silencing under control & IFN gamma stimulation conditions, performed on human primary PBMC-derived macrophages. (C) Relative mRNA & protein expression levels for CCL2, TNF alpha , STAT1, JAK2 & CD36 with GBP1 silencing under control & IFN gamma stimulation conditions, performed on THP-1-differentiated macrophage-like cells. (D) Western blot analysis showing the protein levels of JAK2 & phospho-STAT1 with GBP1 silencing in THP-1 macrophage-like cells at 3 & 6 hr after IFN gamma stimulation. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30303482), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Sortilin forms homodimers on the cell surface of HEK293 cells.A, schematic of FLAG-sortilin & His6-sortilin. FLAG tag & His6 tag were inserted following propeptide & 3 amino acids (Ser78-Ala79-Pro80) in sortilin. SP, signal peptide; PP, propeptide. B, overexpression of FLAG-sortilin & His6-sortilin in HEK293 cells was validated by Western blotting. C & D, detection of binding of FLAG-sortilin & His6-sortilin on the cell surface of HEK293 in TR-FRET assay (C) & HTRF assay (D). Change of FRET signal by expression of His6-sortilin is indicated by percent change (mean ± S.D., three independent experiments). Error bars represent S.D. *, p < 0.05; **, p < 0.01 by t test. IB, immunoblotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29382723), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Western blot analysis of neurofilament proteins. Protein levels of NF-H, phosphorylated NF-H, NF-M & phosphorylated NF-M are enhanced in pEGFP-Peripherin cells, especially at 8 days of NGF induction. However, protein level of NF-L is not influenced in pEGFP-Peripherin cells. The EGFP-Peripherin fusion protein is constantly expressed in pEGFP-Peripherin cells, while not in PC12 cells. The protein level of endogenous peripherin is similar between control PC12 cells & pEGFP-Peripherin cells. Image collected & cropped by CiteAb from the following publication (//jbiomedsci.biomedcentral.com/articles/10.1186/1423-0127-19-8), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Activation of NF-kappa B through NRAGE is specific to the non-canonical BMP pathway. A: 293 cells were transfected with full length NRAGE:EGFP or stimulated with BMP-4 & incubated with or without IKK VII inhibitor, & NF-kappa B activation assessed by luciferase assay. B: 293 cells were transfected with full length NRAGE:EGFP or stimulated with BMP-4 & incubated with or without Tak1 or Tab1 morpholinos, & NF-kappa B activation assessed by luciferase assay. C: Endogenous phosphorylation of IKK-alpha / beta in 293 cells transfected with Tak1 or Tab1 morpholinos prior to 10 ng/ml BMP-4 stimulation for one hour. D: 293 cells were transfected with full length NRAGE:EGFP, incubated with or without Tak1 or Tab1 morpholinos immunoprecipitated with rabbit anti-GFP & western blotted for IKK alpha / beta -phosphorylation & total IKK alpha . E: Western blot of Tak1 & Tab1 proteins in 293 cells with & without morpholinos. Luciferase assays were performed in triplicate & presented as fold increase over renilla. * P < 0.05; vs NRAGE construct or BMP-4 treatment by the two-tailed unpaired Student's t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20100315), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - 16:4(n-3) stimulation increases cPLA2 activity in splenic macrophages. A) Schematic overview of hydrolysis sites of PC to yield metabolites, such as LPC. Splenic macrophages were harvested from murine BALB/c spleens by magnetic bead sorting using F4/80 Abs. Macrophages were incubated with either vehicle or 16:4(n-3) (25 nM) for indicated times. B, C) Fluorescent-based analysis of PLA1 & cPLA2 enzymatic activity revealed a decrease in PLA1 activity (B) & a significant time-dependent increase in cPLA2 activity (C) upon stimulation with 16:4(n-3). Preincubation of splenic macrophages with the GPR120-specific antagonist AH-7614 did not affect 16:4(n-3)-mediated changes in PLA1 activity but was able to prevent the 16:4(n-3)-induced increase in cPLA2 activity. D) In addition, splenic macrophage cell lysates were analyzed by Western blot for phospho-cPLA2. Actin was used as a loading control. E) Treatment of tumor-bearing BALB/c mice with cisplatin (cis) & sCM derived from splenocytes that were incubated with 16:4(n-3) & AACOCF3 did not induce chemoresistance, whereas cotreatment with cisplatin & sCM+ 16:4(n-3) did. Panel B shows grouped results of 2 independent experiments with similar outcomes. Panels C & D show results of 4 independent experiments. Panel E shows grouped results of 2 independent experiments with similar outcomes (n = 8 per group). Data are presented as means ± sem. Statistical significance was determined by 2-tailed Student’s t test (B, C) or 1-way ANOVA (E); all compared with vehicle control (B, C) or with cisplatin alone (E) unless indicated otherwise. *P < 0.05; **P < 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28183801), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - WEGL reduces serum LPS & TLR4-related signalling pathways in HFD mice.Effects of WEGL treatment on serum endotoxin (a) TLR4 protein production (b,c) JNK phosphorylation (d,e) & I kappa B-alpha production (f,g) were examined in the liver & epididymal adipose tissues of chow- & HFD-fed mice as described in Fig. 1. Serum endotoxin (EU ml−1) was determined as mean±s.e.m. using the limulus amebocyte lysate assay kit. Representative immunoblots for target proteins in b-g are shown. Molecular weight markers were indicated as kilodaltons (kDa). Protein levels were normalized to internal controls ( beta -actin or total JNK, T-JNK) & the relative ratio to the Chow group was labelled on the top of immunoblots. Graph bars in a with different letters on top represent statistically significant results (P<0.05) based on Newman–Keuls post hoc one-way ANOVA analysis, whereas bars labelled with the same letter correspond to results that show no statistically significant differences. Where two letters are present on top of the bar, each letter should be compared separately with the letters of other bars to determine whether the results show statistically significant differences. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms8489), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Mutation of Cys783 abolishes dimerization of sortilin.A, schematic of His6-sortilin 10CC+TMD, FLAG-sortilin WT & C783A, & His6-sortilin ICD+TMD WT & C783A. Cysteine 783 was replaced by alanine. SP, signal peptide; PP, propeptide. B, expression vector of His6-sortilin 10CC+TMD was transfected in HEK293 cells. Dimerization of His6-sortilin 10CC+TMD was detected in non-reducing Western blotting with anti-His6 antibody (n = 3). C, sortilin ICD+TMD C783A did not form homodimers in HEK293 cells in the non-reducing Western blotting (n = 3). D & E, C783A decreased sortilin homodimers of low molecular weight in the cells (D) & extracellular vesicles (E) of HEK293 cells in non-reducing Western blotting (n = 3). F & G, 24-h incubation with 2-FPA, an inhibitor of palmitoylation, increased sortilin homodimers of low molecular weight in HEK293 cells stably overexpressing FLAG-sortilin (F) & their extracellular vesicles (G) (n = 3). Monomers & homodimers of high & low molecular weight are abbreviated as MO, D(HMW), & D(LMW), respectively. IB, immunoblotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29382723), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - (a) Cell viability (IC50 values) for a panel of established cancer cell lines as measured by MTS assay following incubation with carfilzomib (Cfz) for 72 h. H727 cells are most resistant to Cfz among 21 tested cell lines. (b) Representative images of H727 cells growing in the presence of Cfz (250 nM) assessed via light microscopy. On day 3 of Cfz treatment, live cell not stained with trypan blue were counted (expressed as % cell viability relative to the vehicle control; no statistically significant difference between Cfz-treated H727 cells & the vehicle control by Student’s t-test, P value > 0.1, n = 3). (c) Immunoblotting results showing no detectable expression of P-glycoprotein (P-gp) in H727 cells. DLD-1 cells with acquired Cfz resistance via P-gp upregulation (DLD-1/CfzR) were used as a positive control. (d) The co-treatment of reversin-121 (7.5 µM, P-gp inhibitor) did not affect the sensitivity of H727 cells to Cfz. The IC50 values did not show statistically significant difference between in the presence & absence of reversin-121 (Student’s t-test). (e) The levels of remaining Cfz in culture media were comparable between H727 & H23 cells (no statistically significant differences, t-tests the Holm-Sidak method to correct for multiple comparisons with alpha = 0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30858500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - (a) Immunoblots showing the expression of cP & iP catalytic subunits in H727 & H23 cells. Immunoblots for additional cell lines displaying differential expression of proteasome catalytic subunits are presented in Supplementary Fig. S2. (b) Heat map displaying differential proteasome activity profiles in H727 & H23 cell lines. Purified human 20S cP & iP were used as controls for individual subunits: 20S cP for beta 5 & beta 1 & 20S iP for beta 5i & beta 1i. The numbers represent hydrolysis rates of respective substrates (RFU/min, mean values derived from three technical replicates) & were converted to color format & clustered by using the program “R” (//www.R-project.org). (c) Remaining catalytic activities of individual proteasome subunits in H727 & H23 cells 4 h after treatment with 20 nM of carfilzomib (left panel) or 20 nM bortezomib (right panel). Data are presented as mean ± SD derived from three technical replicates. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30858500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of insulin on AKT & ERK phosphorylation & cell viability in primary human pancreatic duct cells. Phosphorylated AKT & ERK were measured in primary pancreatic exocrine cultures treated with the indicated concentrations of insulin for 5 minutes (A, B) & 24 hours (C, D) (n =3-4) Fold refers to the fold change of sample relative to control at the same time point. (E) Quantification of automated cell-counting studies employing live-cell imaging of Hoechst-labeled cell cultures over 60 hours. (n =3). (F) Quantification of proliferation by BrdU staining of treated relative to untreated over 3 days (n =4). (G) Quantification of the average number of dying/dead treated cells, propidium iodide (PI) labeled, over 60 hours relative to non-treated cells. (n =3). (H) Human exocrine cells were exposed to 0, 0.2, 2, 20, 200 nM insulin for 3 days. Bright-field images are representative of 3 cultures. (I) Effects of inhibition of RAF1/ERK signalling on PI incorporation with 10 μM GW5074 or AKT signalling with 100 nM Akti1/2 on human primary pancreatic exocrine cell viability (n =3). SF denotes serum free. Repeated Measures ANOVA analyses with Bonferroni’s post-test were performed. *Represents statistical significance of p < 0.05 when compared to DMSO control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25373319), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Liver injury induces intra-nuclear metabolic changes & histone hyper-acetylation. Nuclear (A) acetyl-CoA, (B) lactate, & (C) nHDAC activity in livers harvested 6 h after the injection of either saline or CD95-Ab (n = 3 per group). (D) Western blotting for acetylated histone H3 (H3K9) & H4 (H4K5) on livers harvested 6 h after the injections of either saline or CD95-Ab. Histone H3 & beta -actin were used as loading controls. Each line corresponds to an independent mouse. (E) Affinity purification-liquid chromatography tandem-mass spectrometry of liver proteins enriched by immunoprecipitation with acetyl lysine-specific antibody revealed 13 proteins with fold-change >2 in CD95-Ab vs. saline liver samples (n = 5 per group). (F) Venn diagram including the 751 genes upregulated in livers of mice injected with CD95-Ab & the H3K9Ac ChIP-seq data on eight-week-old mouse livers in ENCODE showing a high degree of overlap (Hypergeometric test; p value: 9.643501e−58). (G) Chart generated using custom annotation scripts shows that response to external stimulus including inflammatory & defense response processes, & cytokine-cytokine receptor interaction pathways were enriched among the 467 genes overlapping between the two groups according to KEGG pathways. Means ± SDs are shown; t test; *p <0.05. Ab, antibody; nHDAC, nuclear histone deacetylase. (This figure appears in colour on the web.) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29580866), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - In vivo inhibition of histone acetylation by garcinol improves survival & reduces injury in acute liver failure. (A) Kaplan-Meier survival curves of mice receiving 20 mg/kg/day of garcinol (n = 8) or saline (n = 5) for 5 days prior to the injection of the CD95-Ab. Means ± SDs are shown. Long-rank test p = 0.0473. (B) Western blotting for histone H3 (H3) & acetylated histone H3 (H3K9) on livers harvested 6 h after the administrations of CD95-Ab. beta -actin was used as a loading control. (C) Densitometric quantifications of H3K9 over H3 from the Western blotting shown in B; t test; *p <0.05. (D) Representative TUNEL (upper panels) & H&E (lower panels) staining of livers harvested 6 h after the administrations of CD95-Ab or saline. (E) Quantifications of TUNEL-positive cells & (F) Necrotic areas on livers of at least three sections from each mouse (at least n = 4 mice per group). Means ± SDs are shown. Likelihood ratio test for generalized linear model: *p = 2.2 × 10−16. ANOVA plus Tukey’s post hoc test; for necrotic area: *p = 2.45 × 10−8. Enzyme activities of PDHC (G) & LDH (H) in nuclear fractions (nPDHC & nLDH) of livers harvested 6 h after CD95-Ab injections in mice treated with garcinol (n = 5) or saline (n = 4). Means ± SDs are shown. ANOVA plus Tukey’s post hoc test: p = 0.11 for nPDHC; p = 0.1 for nLDH. Ab, antibody; A.U., arbitrary units; nLDH, nuclear lactate dehydrogenase; nPDHC, nuclear pyruvate dehydrogenase complex; NS, not statistically significant; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. (This figure appears in colour on the web.) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29580866), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Activation of the mTOR signaling pathway during spheroid formation on chitosan film. (a, Left panel) After 48 hr of seeding on monolayer or with chitosan coating (Spheroid), cell lysates were analyzed using the Human Phospho-MAPK Array kit. (a, Right panel) the spots of phospho-mTOR (S2448) were analyzed by an image analysis system. (b & c) Whole-cell lysates were analyzed by western blotting with specific antibodies against indicated molecules. beta -tubulin & actin were used as the loading control. (d) Immunostaining of p-mTOR (S2448), p-S6 (S235), & p-4EBP1 in 3D spheres. DAPI merged images are shown in the low panel. Scale bar = 50 μM. The results are expressed as the mean ± standard deviation of three independent experiments, which is representative of MSCs from two individuals. **p < 0.01 compared with monolayer cells. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-10708-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Triple AAV Vectors Drive Full-Length Protein Expression in the Pig Retina(A) Western blot (WB) analysis of lysates from Large White pig retina 2 months following subretinal injection of either single or triple AAV2/8 vectors encoding for ED under the control of the PR-specific promoter IRBP. The arrow indicates the full-length protein. (B) WB analysis of truncated products in lysates from Large White pig retina 2 months following subretinal injection of the combinations of triple ED-AAVs. Lysates of HEK293 cells infected with an ED-AAV 1 + 2 + 3 are shown on the left as positive control. The arrows indicate the following products: #A, ED full-length protein; #E, probably from AAV 3. alpha -3xflag, WB with anti-3xflag antibodies; alpha -beta -Actin, WB with anti-beta -Actin antibodies, used as loading control. Neg, lysates of retinas following injection with PBS. The molecular weight ladder is depicted on the left; 200 μg of proteins were loaded. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29292161), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - (a) Immunoblots of proteasome catalytic subunits in H727 cells transfected with siRNA targeting beta 5, beta 2 or beta 5i. (b) The catalytic activity of beta 5 subunit was decreased in H727 cells transfected with siRNA targeting beta 5 compared with H727 cells transfected with scrambled siRNA. (c) Effects of siRNA knockdown of beta 5, beta 5i, or beta 2 on Cfz sensitivity (IC50 values) in H23 & H727 cells. Data are shown as mean ± SD derived from a single non-linear regression based on n = 3 replicates per compound per concentration. (d) Heat map showing proteasome catalytic subunit activity profiles of 6 PI-naïve patient MM samples purchased from Conversant Biologics & AllCells (left). The numbers represent hydrolysis rates of respective substrates (RFU/min, mean values derived from three technical replicates) & were converted to color format & clustered by using the program “R” (//www.R-project.org) (left panel). Carfilzomib (Cfz) cell viability of the same 6 patient MM cells was measured via CellTiter-Glo Luminescent Cell Viability Assay (right panel). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30858500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - 14-3-3 zeta suppresses hedgehog signalling to control the expression of p27Kip1.(a) Measurement of relative luciferase activity from various lengths of the Cdkn1b promoter in control or 14-3-3 zeta -depleted NIH-3T3 cells (n=7–8 per group, *P<0.05 when compared with control cells as assessed by Student's t-test). (b) Transcription factor binding sites within the −939–554 promoter region of Cdkn1b. (c) Hedgehog signalling (Gli-dependent) transcriptional activity in control or si14-3-3 zeta -transfected Shh-light2 cells treated with 100 nM SAG. (n=4 per group, *P<0.05 when compared to control cells, as assessed by Student's t-test). (d) Expression levels of various components of the hedgehog signalling pathway in differentiating control & 14-3-3 zeta -depleted 3T3-L1 cells, as measured by RNA-seq (n=4 per group, *P<0.05 when compared to control cells, as assessed by Student's t-test). (e) Immunoblotting of full length (∼170 kDa) & processed (∼80 kDa) Gli3 in 14-3-3 zeta -depleted 3T3-L1 cells treated with MDI (representative of n=3 experiments). (f,g) Co-immunoprecipitation of endogenous (f) 14-3-3 zeta or (g) Gli3 from lysates of 3T3-L1 cells treated with MDI for 24 h (representative of n=3 experiments) (h) ChIP-qPCR analysis of Gli3 occupancy on the Cdkn1b promoter in 3T3-L1 cells treated with MDI for 24 h (n=4 per group, *P<0.05, as assessed by Student's t-test). (i,j) Co-transfection of siRNA against 14-3-3 zeta & Gli3 was used to examine whether knockdown of both proteins could restore differentiation, as determined by Ppar gamma expression (i) or Oil Red-O staining (j) (n=4 independent experiments). Error bars represent s.e.m. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26220403), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Silencing MFN1 or MFN2 reveals their distinct roles in DENV infection.(A & B) RT-qPCR (A) & western blot (B) validation of A549 cells stably expressing shRNA targeting LacZ, MFN1 or MFN2. Data in panels A & C-F are mean ± SD (n = 3 per group) & were compared by two-tailed Student’s t test. Quantification in panel B is the relative ratio of the indicated protein to actin expression. (C) LDH release assay of shLacZ-, shMFN1- or shMFN2-expressing A549 cells infected with DENV serotype 2 (moi 10) for the indicated time. (D) DENV plaque-forming assay of shLacZ-, shMFN1- or shMFN2-expressing A549 cells infected with DENV (moi 0.1) for 24 or 48 h. (E) Analysis of antiviral activity in culture media from indicated cells with DENV infection (moi 5, 48 h) against dSinF-Luc/2A virus. (F) RT-qPCR analysis of IFN beta mRNA level in DENV infected (moi 10) A549-shLacZ, -shMFN1, & -shMFN2 cells for the indicated time. (G) Western blot analysis of DENV infected (moi 10) A549 cells expressing shLacZ, shMFN1 or shMFN2. The relative ratios of band intensity were quantified as indicated. (H) Flow cytometry with JC-1 staining of shLacZ-, shMFN1 or shMFN2-expressing A549 cells infected with DENV (moi 5) for 30 h. Decreased red/green fluorescence ratio of JC-1 represents disrupted MMP. CCCP, carbonyl cyanide m-chlorophenyl hydrazone, an ionophore that disrupts MMP. (I) Plaque forming assay of vesicular stomatitis virus (VSV) by using A549 cells expressing shLacZ, shMFN1 or shMFN2 as indicated. pfu, plaque-forming unit. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1005350), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on IL-18 gene expression & secretion in human macrophages.Cells were pre-treated with various concentrations (1 or 2%) of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & ATP (5 mM) for 1 h. (a) The mRNA expression levels of IL-18 were determined by RT-PCR analysis. (b) IL-18 mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) The amount of IL-18 in cell culture supernatants was detected by ELISA. (d) The presence of IL-18 in cell lysates & cell culture supernatants were analyzed by Western blot analysis. Data are presented as means ± SE of three experiments performed in duplicate. #P < 0.01 versus untreated cells. *P < 0.01 versus HSM-untreated control (ethanol) cells. †P < 0.05 versus HSM (1%) treated cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Wnt effect on AChR clustering requires interaction with MuSK. A, Wnt9a & Wnt11, but not Wnt7a, bind MuSK in vitro. Flag-Wnts immobilized beads were incubated with MuSKect-Myc. Bound proteins were isolated, resolved by SDS-PAGE & blotted with antibodies against Myc & Flag. B, Wnt11 binds CRD of MuSK. Flag-Wnt11 immobilized beads were incubated with MuSKect, MuSKect delta CRD or MuSKCRD. Interactions were assayed as in A. *, p < 0.05, n = 3. C, Wnt-induced AChR clustering is rescued by MuSK, but not MuSK delta CRD, in MuSK-/- cells. MuSK-/- cells were transfected without or with respective constructs & resulting myotubes (identified by GFP encoded by the expression construct) were assayed for AChR cluster formation in response to agrin or Wnts as in Figure 1. Arrows, AChR clusters. *, p < 0.01, Student's t test. D, Wnt9a & Wnt11 induce MuSK tyrosine phosphorylation. C2C12 myotubes were treated by agrin or Wnts for 1 h. MuSK was purified by immunoprecipitation & probed with the 4G10 antibody. E, Wnt9a & Wnt11 induce MuSK dimerization. Flag-MuSK & MuSK-Myc were transfected into C2C12 myoblasts & resulting myotubes were treated by Wnts, agrin or control media for 1 h. Flag-MuSK was precipitated by Flag antibody & associated MuSK-Myc was determined by anti-Myc antibody. Band intensity of immunoblot was analyzed by ImageJ software. *, p < 0.01, one-way ANOVA with Student's t test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/22309736), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Amiodarone enhanced liver growth & hepatocyte proliferation in the liver regeneration after PHx.Wild-type mice were intraperitoneally injected with vehicle (Veh), amiodarone (AD), or chloroquine (CQ) at 0.5 h before PHx or sham operation & then once per day until 168 h. Liver tissues were harvested at 0–168 h after surgery. Liver-to-body-weight ratios were calculated (A). Liver sections at 24 h after the sham operation, PHx with Veh, AD, or CQ were stained with H&E; original magnification, 400X (B). Representative immunohistochemical staining of Ki67 is shown (C). The percentage of Ki67-positive nuclei in hepatocyte was counted under low-power fields (200 ×) in 15 random sections from at least six different mice (D). Liver tissues were harvested at 0 h, 12 h, 24 h, or 48 h after surgery, & the tissue extracts were analyzed for PCNA, cyclin A, B, D1, E, p21, TGF-beta 1, & beta -actin protein by Western blotting (E,F). The values are shown as the mean ± SD in the bar graph & compared by Student’s t test. #P < 0.05 versus vehicle-treated PHx. Scale bar, 25 μm in B, 50 μm in C (n = 6). Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep15807), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Cell death & DNA damage in response to treatment in A549/p53 dominant-negative cells(A) Confirmation of stable clones was done by western blot analysis using p53 specific antibody. (B) MTT assay for cisplatin shows that the mutant cell line is resistant to the drug as compared p54-wt A549 cells. (C) Confocal images of A549/p53 dominant negative cells shows an elevated level of DNA damage in combination than single agent treatment. Increased accumulation of gamma -H2AX indicates DNA damage. Nucleus= Blue (DAPI), F-actin= Red (TRITC), gamma -H2AX= Green (FITC). (D) Western blot analysis shows a caspase mediated cell death in A549/p53 mut cells in response to treatment. (E) AnnexinV-PI dual staining & flow cytometry analysis shows significantly higher apoptosis in combination treatment than low dose cisplatin & scriptaid. Q1=necrotic population, Q2=late phase apoptosis, Q3=live cells, Q4= early phase apoptosis. C= control, L=2μg/ml cisplatin, S1= 1μg/ml scriptaid. * indicates p < 0.05. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.12378), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Sortilin forms homodimers in the extracellular & intracellular domains with intermolecular disulfide bonds in HEK293 cells.A, schematic of FLAG-sortilin Full, ECD+TMD, & ICD+TMD. SP, signal peptide; PP, propeptide. B & C, protein expression of FLAG-sortilin Full, ECD+TMD, & ICD+TMD was validated in reducing (B) & non-reducing (C) Western blotting using anti-FLAG antibody. FLAG-sortilin Full & ECD+TMD form homodimers & multimers. Empty vector was used as a control. D, HEK293 cells transiently overexpressing FLAG-sortilin Full or ECD+TMD were treated with a cross-linker, BS3, & the cell lysates were used for reducing Western blotting with anti-FLAG antibody, showing dimerization of sortilin Full & ECD+TMD (n = 3). E, HEK293 cells stably overexpressing FLAG-sortilin ICD+TMD (FLAG-sortilin ICD+TMD HEK293 cells) were incubated with DMSO (Control), 20 μmol/liter MG-132 (MG) or 10 μmol/liter chloroquine (Chlo) for 7 h, & then reducing Western blotting was performed using anti-sortilin antibody. MG-132 increased the protein expression of FLAG-sortilin ICD+TMD, but chloroquine did not (n = 3). F, FLAG-sortilin ICD+TMD HEK293 cells were incubated with DMSO or MG-132 (2–20 μmol/liter) for 7 or 24 h. MG-132 increased FLAG-sortilin ICD+TMD in a time- & concentration-dependent manner (n = 3). G, following 16-h incubation of HEK293 cells (Control) or FLAG-sortilin ICD+TMD HEK293 cells (ICD+TMD) with MG-132 (5 μmol/liter) & immunoprecipitation with anti-FLAG antibody, non-reducing Western blotting showed dimerization of sortilin ICD+TMD using anti-sortilin antibody (n = 3). Monomers, homodimers, & multimers are abbreviated as MO, D, & MU, respectively. IB, immunoblotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29382723), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Defects in adipogenesis due to 14-3-3 zeta depletion are due to cell cycle arrest & up-regulation of Cdkn1b/p27Kip1.(a–d) Analysis by flow cytometry (a) of control or si14-3-3 zeta -transfected 3T3-L1 cells at different stages of the cell cycle at 0 (b), 24 (c) & 48 (d) hours following differentiation (n=4 independent experiments; *P<0.05 when compared with siCon-transfected cells, assessed by Student's t-test). (e) Analysis of various cell cycle regulatory genes by RNA-Seq from siCon- or si14-3-3 zeta transfected 3T3-L1 adipocytes (n=4 per goup, *P<0.05 when compared with siCon cells at the same time point, assessed by Student's t-test). (f) Immunoblotting of p27Kip1 from lysates of differentiating of siCon- or si14-3-3 zeta -3T3-L1 adipocytes (n=4 experiments). (g,h) Co-transfection of siRNA against 14-3-3 zeta & p27Kip1 was used to examine whether knockdown of both proteins could restore differentiation, as determined by Ppar gamma expression (g) or Oil Red-O staining (h). (n=4 independent experiments). Error bars represent s.e.m. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26220403), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on IL-1 beta gene expression & secretion in human macrophages.Cells were pre-treated with either 1 or 2% of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & with ATP (5 mM) for 1 h. (a) The mRNA expression levels of IL-1 beta were determined by RT-PCR analysis. (b) IL-1 beta mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) The amount of IL-1 beta in cell culture supernatants was detected by ELISA. (d) The presence of IL-1 beta in cell lysates & cell culture supernatants were analyzed by Western blot analysis. Data are presented as means ± SE of three experiments performed in duplicate. #P < 0.01 versus untreated cells. *P < 0.01 versus HSM-untreated (ethanol-treated) control cells. †P < 0.05 versus HSM (1%) treated cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - (A) shows western blots of nuclear extracts of mCCD cells treated with 100 μmol/L CoCl2. Treatment with CoCl2 increased stabilization of HIF1 alpha in the nucleus of the cells. GAPDH levels are shown as loading controls. (B) shows western blots of whole cell extracts of mCCD cells treated with 100 μmol/L CoCl2 for 4 h. As shown in the images, treatment with CoCl2 increased the EPO production by cells. Beta actin levels are shown as loading controls. (C) shows western blots of whole cell extracts of mCCD cells treated with 100 μmol/L CoCl2 for 4 h & chronic fluid flow. As shown in the images, treatments decreased ZO‐1 production by cells independently of fluid flow stimulation. Beta actin levels are shown as loading controls. (D) shows western blots of whole cell extracts of mCCD cells treated with 100 μmol/L CoCl2 for 4 h & chronic fluid flow. As shown in the images, treatments decreased NaKATPase production by cells independently of fluid flow stimulation. Beta actin levels are shown as loading controls. (E) Densitometric analysis of Western blots presented in (A–D). HIF1 alpha level: P = 0.004 (N = 3), t‐test, showing significant nuclear stabilization of HIF1 alpha . EPO level: increases slightly though not statistically significant (N = 3, P > 0.05, One‐way ANOVA). ZO‐1 level: P < 0.0001 (N = 4), One‐way ANOVA, showing significant decrease in ZO‐1 levels, independent of fluid flow. NaKATPase alpha 1 subunit level: P = 0.001 (N = 3), One‐way ANOVA, showing significant decrease in NaKATPase alpha 1 subunit levels, largely independent of fluid flow. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29263117), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - MFN1 & MFN2 manipulate MAVS-triggered signaling differently.(A & B) Western blot (A) & RT-qPCR (B) analysis of A549+on/myc-MAVS cells with stably expressing shRNA targeting the indicated genes after 48 h of Dox treatment. RT-qPCR data are mean ± SD (n = 3 per group) & were compared by two-tailed Student’s t test. (C) Dual-luciferase activity analysis of A549 cells cotransfected with the indicated MFNs (cloned in pcDNA3.1 vector; 0.5 μg), stimulator or control (Flag-MAVS/pcDNA3 or Flag-GFP/pcDNA3; 0.4 μg), IRF3/pCR3.1 (0.3 μg), p125-Luc (0.2 μg) & pRL-TK (0.1 μg) for 24 h. The firefly luciferase activity (p125-Luc) was normalized to that of renilla luciferase (pRL-TK) & the relative luciferase activities are shown. Data are mean ± SD (n = 3 per group) & were compared by two-tailed Student’s t test. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1005350), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on inflammasome components & caspase-4 activation in human macrophages.Cells were pretreated with various concentrations (1 or 2%) of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & ATP (5 mM) for 1 h. (a) The mRNA expression levels of ASC, NLRP1, NLRP3 & caspase-4 were determined by RT-PCR, using beta -actin as the internal control. (b) ASC, NLRP1, NLRP3 & caspase-4 mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) Cell lysates were analyzed by Western blot analysis using specific anti-ASC, anti-NLRP1 & anti-NLRP3 antibodies. (d) Cell lysates were analyzed for protein levels of caspase-4 by Western blot analysis. beta -actin was used as an internal control. Data are presented as means ± SE of three experiments preformed in duplicate. #P < 0.01 versus untreated cells. versus HSM-untreated control (ethanol) cells. *P < 0.01 versus HSM-untreated control (ethanol) cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Proteasome inhibitors restored the expression of MFN2 & induced apoptosis under hypoxic conditions. HPASMCs were exposed to hypoxic conditions & were treated with bortezomib & marizomib for 24 h. The expression of MFN2, pro-PARP1, & cleaved PARP1 were detected by Western blotting. The expression of actin was used as a loading control. Bortezomib (A) & marizomib (B) restored the expression of MFN2-inhibited by hypoxia, inhibited the levels of pro-PARP1, & induced the level of cleaved PARP1. One-way ANOVA was used to determine the differences between the experimental & vehicle control (DMSO) groups & the Newman–Keuls Multiple Comparison Test was used as a post hoc test following ANOVA. The data represent means ±standard error of three independent experiments. * p < 0.05 versus normoxic group. # p < 0.05 versus hypoxic control group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35453623), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Ad.5-TCTV induces ER stress & apoptosis in cancer cells. (A) Breast cancer cells (MDA-MB-231 & SUM-159) treated with 25 pfu of Ad.5-E1A or the indicated pfu of Ad.5-CTV or Ad.5-TCTV for 72 h. Cells were then collected & Western blotting analysis was performed for signaling molecules using specific antibodies & beta -actin served as a loading control. (B) Breast cancer cells were cultured in 8-well chamber slides & infected with 25 pfu of Ad.5-E1A, Ad.5-CTV, or Ad.5-TCTV for 72 h. Cells were fixed & TUNEL assays were performed. Data presented as TUNEL positive cells in a defined microscopic field as compared with un-treated control cells. (C) Prostate cancer cells (DU-145 & PC-3) were treated as described in A, cells were then collected & Western blotting analysis was performed for signaling molecules using specific antibodies & beta -actin served as a loading control. (D) Prostate cancer cells were treated as described in B & cells were fixed & TUNEL assays were performed. Data presented as TUNEL positive cells in a defined microscopic field as compared with un-treated control cells. Scale bar: 200 μM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33670594), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Pyruvate dehydrogenase kinase 1 (PDK 1) regulates DRP1 mediated mitochondrial fission & glycolytic phenotype in BRAFV600E cells. (A, B) Western blot & representative confocal micrographs (2.5x zoom, 60x original magnification) of si-PDK1 silenced BRAFV600E CRC cells, showing a reduction in both pDRP1S616 levels as well as mitochondrial fission after PDK1 knockdown, 30-40 cells were quantified (n=3, mean± SEM), red= mitochondria, CMX Ros stained & blue= Nuclei, DAPI stained, statistical analysis was done by two way ANOVA, followed by Bonferroni post-tests comparing replicate means by column with corresponding p values shown. (C, D) Genetic & pharmacologic knockdown of PDK1 in BRAFV600E CRC cells leads to a reduction in cell proliferation (n= 4, mean± SEM) & clonogenic rates in soft agar (n=3, mean± SEM) respectively; statistical analysis for proliferation was done by 2-way ANOVA followed by Bonferroni post-tests comparing replicate means by row with corresponding p values shown, & statistical analysis for soft agar clonogenic assay was done by unpaired t-test followed by Welch’s correction assuming unequal variance in means, corresponding p values shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33738242), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Expression of Secretagogin in different regions of human & rat brain. Tissues from different brain areas were analyzed for relative Secretagogin gene & protein expression by RT-qPCR & Western blotting. (A) Human post-mortem tissues from three individuals were analyzed with regard to the following specific brain regions: cerebellum, frontal cortex, parietal cortex, hippocampus, thalamus, & olfactory bulb. Western blot analysis was performed from tissue of white matter, thalamus, hypothalamus, hippocampus, stem ganglia, cerebellum, frontal cortex, parietal cortex, & occipital cortex. Equal protein loading was verified by staining with beta -actin antibody. (B) Rat brain tissues from three adult rats were dissected & analyzed from cerebellum, frontal cortex, parietal cortex, hippocampus, striatum, & olfactory bulb. A representative Western blot of equal amounts of protein from cerebellum, frontal cortex, parietal cortex, hippocampus, striatum, & olfactory bulb is shown on the right. beta -actin immunostaining was again used as loading control. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/article/10.3389/fnmol.2012.00084/abstract), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Lack of HIF-P4H-1 increases the amount of p53. (a) Western blot analysis of p53 in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (b,c) Western blot analysis of p53 in Hif-p4h-1 & scrambled (Scrm) siRNA transfected HEK293 cells (b) & wt MEFs treated with 50 µM FG4497 for 24 h (c). (d) qPCR analysis of p53 mRNA in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (e) Analysis of p53 protein turnover rate. Hif-p4h-1−/− & wt MEFs were treated with 200 μg/ml of cycloheximide for the indicated time points & whole cell lysates were blotted for p53. (f) Western blot analysis of MDM2 in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h. (g) Western blot analysis of p53 & MDM2 in wt MEFs treated with 10 μM nutlin-3a for 24 h with or without overexpression of human HIF-P4H-1 (OE). (h,i) Western blot analysis of HIF1 alpha in wt & Hif-p4h-1−/− MEFs cultured in 21% or 1% O2 for 24 h (h) & in scrambled & Hif-p4h-1 siRNA transfected HEK293 cells (i). (j) Western blot analysis of ubiquitination of p53 in Hif-p4h-1 & scrambled siRNA transfected HEK293 cells. The cells were transfected with HA-ubiquitin & endogenous p53 was immunoprecipitated followed by Western blotting with anti-HA & anti-P53 antibodies. (k) Western blot analysis of p53 in Hif-p4h-1−/− & wt MEFs were treated with or without 10 μM MG132 for 5 h. Data are presented as representative Western blots & as mean ± s.d., n = at least 3 individual MEF isolates or experiments. *P < 0.05, **P < 0.01 & ***P < 0.001, two-tailed Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig. 5. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-17376-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Lack of HIF-P4H-1 suppresses NF-kappa B activity. (a) Western blot analysis of Rel-A in wt & Hif-p4h-1−/− MEFs subjected to 21% or 1% O2 for 24 h. (b–d) Analysis of NF-kappa B luciferase reporter activity in wt & Hif-p4h-1−/− MEFs (b), in Hif-p4h-1 & scrambled (Scrm) siRNA transfected HEK293 cells (c) & in MDA-MB-231 cells treated with 50 µM FG4497 for 6 h (d). Cells were harvested 48 h after the NF-kappa B luciferase reporter plasmid transfection. siRNA transfection was performed 24 h before the NF-kappa B luciferase reporter plasmid transfection. FG4497 was added 6 h before the cell harvest. (e) Western blot analysis of Rel-A, p-Rel-A, I kappa B alpha , p105 & p50 in wt & Hif-p4h-1−/− MEFs treated with or without 200 ng/ml LPS for 12 h. (f,g) Analysis of Rel-A & p-Rel-A by Western blotting (f) & NF-kappa B luciferase reporter activity (g) in wt MEFs treated with or without 200 ng/ml LPS & 50 µM FG4497 for 6 h. (h,i) Analysis of Rel-A & p-Rel-A by Western blotting (h) & NF-kappa B luciferase reporter activity (i) in Hif-p4h-1−/− MEFs transfected with empty vector (EV) or a vector encoding V5-tagged human recombinant HIF-P4H-1 (OE) & treated with or without 200 ng/ml LPS for 12 h. (j) qPCR analysis of Rel-A mRNA in Hif-p4h-1−/− & wt MEFs treated with 200 ng/ml of LPS for 12 h. (k) Western blot analysis of Rel-A & p-Rel-A in cytosolic & nuclear fractions of Hif-p4h-1−/− & wt MEFs. (l) NF-kappa B luciferase reporter activity in HIF1A−/− HCT116 cells treated with FG4497 or transfected with HIF-P4H-1 siRNA. Data are presented as representative Western blots & as mean ± s.d., n = 3–4 individual MEF isolates or experiments. *P < 0.05, **P < 0.01 & ***P < 0.001, two-tailed Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig. 5. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-17376-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - The cleaved MFN fragments were further degraded by host proteasome machinery.(A) Western blot analysis of A549+on/MFN1 (lane 1–6) & /MFN2 (lane 7–12) cells with Dox treatment (all lanes) & DENV infection (lane 3–6 & 9–12). The cells were infected with DENV serotype 2 (moi 5) for 24 h, then incubated in culture media containing Dox (1 μg/ml) with (lanes 5–6 & 11–12) or without MG132 (0.5 μM) for 16 h. (B) Western blot analysis of four different serotypes DENV-infected A549 cells incubated with MG132-containing medium. (C) Western blot analysis of endogenous MFN1 & 2 cleavage in A549 stable cells overexpressing WT or protease-dead mutant (S135A) of DENV NS2B3 with or without 18-h treatment with MG132 (0.5 μM). Filled arrow: full-length; open arrow: cleaved product; star: non-specific band. The longer exposure images for endogenous MFN1 & 2 signals are also shown in panels B & C. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1005350), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Zinc-finger motifs of ZAP are required for JEV RNA binding & its antiviral activity.(A) Schematic representation of human ZAP isoforms (ZAP-L, 902 a.a. & ZAP-S, 699 a.a.). The four tandem CCCH-type zinc-finger (ZF) motifs within the N-terminus of ZAP are shown as solid black boxes. Deletion of the four ZFs (a.a. 73–193) are indicated with a dashed line. (B) 293T/17 cells infected with JEV (MOI = 5) for 16 h were transfected with plasmids expressing EGFP, WT or ZF-deleted ZAP-L-V5 & ZAP-S-V5 for additional 24 h. The viral RNA bound with V5-tagged proteins was pulled down by anti-V5 agarose affinity gel & amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input viral RNA in JEV-infected cells (lower panel). Western blot analysis of the immunoprecipitated ZAP-L & ZAP-S (WT & del4ZFs) (upper panel). (C-E) The indicated cells were infected with JEV (MOI = 5) for 10 h. Cell lysates, total RNA, & culture supernatants were determined for the indicated proteins by western blot (C), viral RNA by RT-PCR (D), & viral titer by plaque assay (E). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. *** P≤0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30016363), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - CD95-induced senescence is mediated by low-level canonical caspase signaling. (a) CRC29 colonospheres were exposed once (cycle 1) or chronically exposed to CD95L (10 ng/ml) for 2 weeks (cycle 2), & collected at the indicated time points. Cells were collected & analyzed for the presence of activated (cleaved) caspase-3 by western blotting. (b–c) CRC29 colonospheres were exposed to CD95L (5 ng/ml) in the presence or absence of zVAD (25 μM) for 1 or 7 days & were then processed for imunofluorescence analysis of caspase-3 activation (b) & DNA damage ( gamma -H2AX) (c) An overview of the significance of all comparisons is provided in Supplementary Table S1. (d) Colonspheres were exposed to CD95L (10 ng/ml) for 24 h in the presence or absence of zVAD (25 μM) followed by western blot analysis of caspase cleavage & iCAD processing. (e & f) CRC29 colonospheres were exposed to increasing concentrations of CD95L as indicated in the presence or absence of zVAD (25 μM), after which SA-beta GAL (e) & clone-forming capacity (f) were assessed at 7 or 14 days, respectively. The asterisks indicate significant differences (ordinary one-way ANOVA) (****P<0.00001; **P<0.001). An overview of the significance of all comparisons is provided in Supplementary Table S1 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28300842), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - mRNA & protein expression in Subject 1 & control lymphoblast cells. A. Scatter graph of mRNA expression of EXOC6, RPJB and HES1in lymphoblasts. Values for EXOC6, RPJB and HES1 mRNA expression were normalized to the corresponding beta -actin mRNA levels. The results derived from at least three sets of samples & the mean level of each sample is represented in the graph, horizontal bar is an average value between three controls. The difference between the study sample & the average of the controls has been evaluated by student t-test (*, p < 0.05; **, P < 0.01). B. RBPJ protein expression. C. HES1 protein expression. C1-C5 indicate control samples from the lymphoblasts, S1 indicate study sample from Subject1. Cell lysates were analyzed by SDS-PAGE & immunoblotting with membrane probed for RBPJ, HES1 & beta -actin, Values for RBPJ & HES1 expression were normalized to the corresponding beta -actin levels. The results derived from at least three sets of samples & the mean value of each sample is represented in the bar graph. The difference between Subject1 & the average of three controls has been evaluated by student t-test (*, p < 0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23837398), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Ketogenic diet induces anti-inflammatory IL-4 & Arginase-1 expression in D2 retina & ON. a Bar graph showing Il-4 mRNA normalized to Hprt mRNA by qRT-PCR in untreated (Unt) & keto D2 retina, *p = 0.0264; n = 8 samples per group. b, c IL-4 protein levels in the retina (b), **p = 0.0014, & ON (c), *p = 0.0121, analyzed by ELISA; n = 8 samples per group. dArg1 mRNA levels in retina normalized by Hprt mRNA levels, *p = 0.0368; n = 8 samples per group. e Immunohistochemical analysis of Arginase-1 (green) in the untreated & keto D2 retina & ON. Iba1 (red) labels microglia & DAPI stains nuclei. f, g Percentage of mean fluorescence of Arginase-1 in inner retina (f), ***p = 0.0001, & in the ROI of the proximal ON (g), ***p = 0.0001; five retinas & ON from each group were analyzed, with five sections from each individual retina & ON. h Western blot analysis of Arginase-1 protein in the retina. i Quantification by densitometry of Arginase-1 levels normalized to beta -actin levels, ***p = 0.0001; n = 3 blots per group, each with independent samples. j Arginase-1 protein in the ON normalized to beta -actin levels, as determined by capillary electrophoresis, **p = 0.0006; n = 3 ON per group. All bar graphs are presented as the mean ± SEM, analyzed by two-tailed unpaired t test. Scale bar, 20 μm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30424795), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Substituting the transmembrane domain of sortilin with the corresponding domain of CD43 does not decrease the dimeric form of sortilin.A, schematic of FLAG-sortilin wildtype (WT) & FLAG-sortilin CD43-TMD. The transmembrane domain of sortilin was replaced with that of CD43. SP, signal peptide; PP, propeptide. B, FLAG-sortilin WT & FLAG-sortilin CD43-TMD were transiently overexpressed in HEK293 cells, & non-reducing Western blotting was carried out using cell lysate with anti-FLAG antibody (n = 3). Monomers, homodimers, & multimers are abbreviated as MO, D, & MU, respectively. C & D, His6-sortilin WT or His6-sortilin CD43-TMD was transiently overexpressed in HEK293 cells stably overexpressing FLAG-sortilin, & immunoprecipitation was performed using anti-FLAG M2 antibody. Western blotting was carried out using whole-cell lysates (C) & immunoprecipitants (D). His6-sortilin CD43-TMD coprecipitated with FLAG-sortilin as well as His6-sortilin WT. Arrows, sortilin wildtype or sortilin CD43-TMD (n = 3). E, in FLAG-sortilin HEK293 cells or HEK293 cells, His6-sortilin CD43-TMD was overexpressed. The cells were subjected to TR-FRET assay. Change of FRET signal by expression of His6-sortilin WT or CD43-TMD is indicated by percent change (mean ± S.D., n = 4, one independent experiment). Error bars represent S.D. *, p < 0.05; **, p < 0.01 by t test. F & G, in FLAG-sortilin HEK293 cells, His6-sortilin WT or His6-sortilin CD43-TMD were overexpressed. The cell lysates were subjected to non-reducing Western blotting with anti-FLAG antibody (F) & anti-His6 antibody (G), demonstrating that substituting the transmembrane domain of sortilin with that of CD43 did not decrease dimerization (n = 3). IB, immunoblotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29382723), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - The expression of HO-1 is higher in ALI/ARDS-developing mice compared to HP-developing mice. (a) Representative images of lung sections subject to immunohistochemistry for detection of HO-1 protein (brown), counterstained with hematoxylin. The graph represents the quantification of protein expression of HO-1 by immunohistochemistry on the 7th day after infection (DAI). Dashed line represents the average of values from noninfected mice. (Mann–Whitney test, n = 10, ∗∗p ≤ 0.01). (b) Immunoblot of HO-1 & beta actin control (left). Protein levels of HO-1 measured by immunoblot densitometry (right). Values are expressed in HO-1 band densities adjusted by the beta actin control. (c) Expression of HO-1 mRNA levels in lungs of ALI/ARDS-developing mice & HP-developing mice (unpaired t-test, n = 28, ∗p ≤ 0.05). (d) Values of HO-1 in lung cell lysates of ALI/ARDS versus HP-developing mice (Mann–Whitney test, n = 8, ∗p ≤ 0.05). (e) Protein levels of HO-1 in the serum of ALI/ARDS-developing mice & HP-developing mice (Mann–Whitney test, n = 9, ∗p ≤ 0.05). (f) Bilirubin levels in the serum of ALI/ARDS & HP infected mice. Bilirubin levels are significantly higher in ALI/ARDS than in noninfected mice (one-way ANOVA with Bonferroni's multiple comparison test n = 38, #p ≤ 0.05). The dashed lines represent the average values of noninfected mice (minimum n = 3). In graphs ((b) & (e)) the values of noninfected mice were equal or less than 0. In graphs with fold increase, the values of ALI/ARDS-developing & HP-developing mice are compared to the average values of noninfected mice. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27974865), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Analysis of NLRP3, IL-1 beta , & ARRB2 expression in untreated (Unt) & Keto D2 & D2G retina & ON. a, bNlrp3 (a), *p = 0.0219, & Il-1 beta (b), *p = 0.0151 mRNA levels normalized to Hprt mRNA levels determined by qRT-PCR; n = 8 samples per group. c Western blot analysis of NLRP3 & IL-1 beta protein levels in the retina. d, e Quantification by densitometry of NLRP3 (d), ***p = 0.0001, ***p = 0.0001, & mature IL-1 beta (e), ***p = 0.0001, ***p = 0.0001 protein levels normalized to beta -actin levels; n = 3 blots per group, each with independent samples. f IL-1 beta protein levels in the ON analyzed by ELISA, **p = 0.0092; n = 8 samples per group. g Western blot analysis of ARRB2 protein levels in the retina. h Quantification by densitometry of ARRB2 protein normalized to beta -actin; n = 3 blots per group, each with independent samples. i Capillary tube electrophoresis of ARRB2 protein levels in the optic nerve normalized to beta -actin ***p = 0.0001, ***p = 0.0001; n = 3 ON per group. All bar graphs are presented as the mean ± SEM, n = 5–9, analyzed by two-tailed unpaired t test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30424795), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Sortilin S316E & sortilin wp increase dimerization in HEK293 cells, & the addition of propeptide decreases dimerization in the extracellular vesicles of FLAG-sortilin HEK293 cells.A, schematic of FLAG-sortilin WT, S316E, & wp. Serine 316 was replaced by glutamic acid in FLAG-sortilin S316E. Propeptide was removed in FLAG-sortilin wp. SP, signal peptide; PP, propeptide. B, S316E increased dimerization of sortilin in HEK293 cells (n = 3). C, removal of propeptide increased dimerization of sortilin in HEK293 cells (n = 3). D & E, addition of propeptide (100 nmol/liter) decreased dimerization of sortilin in the extracellular vesicles of FLAG-sortilin HEK293 cells (E), whereas a decrease in the cells was not observed (D) (n = 2). Monomers & homodimers of high & low molecular weight are abbreviated as MO, D(HMW), & D(LMW), respectively. Vec, vector; IB, immunoblotting. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29382723), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Chronic CD95 stimulation reduces colony-forming capacity in human colonospheres. (a) Human colonosphere cultures isolated from the tumors of nine distinct colon cancer patients were pretreated with FC control or FC-CD95L (10 ng/ml) for 2 weeks, seeded as single cells in Matrigel & cultured continuously in the absence or presence of FC-CD95L. Colony formation was scored after 2 weeks. The experiments were performed multiple times with similar results (L145: n=4, L146: n=4, L167: n=3, CRC47 n=4, CRC29 n=3, L193 n=2, CRC26 n=1, L169 n=4, CR16 n=3). One representative experiment is shown (triplicate values). (b) The expression of CD95 was measured with FACS (CH11, left panel) & with western blotting (middle panel; asterisks indicate non-specific bands). The FACS results are plotted as the percentage CH11-positive cells over control for each cell line. Averaged FACS values were then plotted against the percentage growth inhibition (right panel). The Pearson correlation & the accompanying P-value are shown in the inset. (c) CRC29 cells were cultured in the presence of FC control or FC-CD95L for 2 weeks & were then processed for FACS analysis of DNA content using PI. (d) CRC29 cells were cultured in the presence of control or CD95L for 2 weeks, labeled with CFSE, & subsequently plated in Matrigel as single cells. Cells were collected from the Matrigel & analyzed for maintenance of the fluorescent signal by FACS at days 2, 4, & 7 after plating. (e) The experiment was performed as in a, comparing a 2-day treatment to chronic stimulation with FC-CD95L during colony formation Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28300842), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - JEV NS5 interacts with mitochondrial trifunctional protein (MTP), the enzyme complex involved in LCFA beta -oxidation.(A) Western blot analysis of protein levels of the indicated proteins in A549 cells with JEV infection (MOI = 10) or NS5-Flag overexpression. (B) Western blot analysis of V5-tag, Flag-tag, & actin in HEK293T cells co-transfected with HADH alpha -V5-His plus the indicated plasmids expressing Flag-tagged JEV viral proteins for 24 h, then immunoprecipitated with anti-Flag affinity gel. WCL, whole-cell lysates. (C) Immunoprecipitation (IP) analysis with control IgG or anti-Flag affinity gel in A549, GFP-A549 & NS5-Flag-A549 cells. The protein bands identified as HADH alpha & HADH beta are indicated by arrows. (D) IP with anti-Flag affinity gel & Western blot analysis with the indicated antibodies in HEK293T cells co-transfected with vector control or NS5-Flag plus HADH alpha -V5-His or HADH beta -HA for 24 h. (E) IP analysis with nickel beads & Western blot analysis with the indicated antibodies in HEK293T cells adsorbed with JEV for 3 h, then transfected with vector control, HADH alpha -V5-His or HADH beta -V5-His for 24 h. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1004750), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Celecoxib induced cell cycle arrest at the G2 phase in CGCCA cells.CGCCA cells were treated with the indicated concentration of celecoxib for 1 day, & the cell cycle distribution was analysed. (A & B) After 1 day of celecoxib treatment, CGCCA cells were analysed by flow cytometry to determine cell cycle distribution. (C) Treated CGCCA cells were stained with PI to show condensed chromatin (white arrow) in M-phase cells. In the right lower panel, the (+) & (–) are examples of cells with & without condensed chromatin, respectively. (D) Ratio of CGCCA cells at M-phase. (E) Expression levels of Cdc25C & CDK1 in CGCCA cells after 1 day of treatment with the indicated concentrations of celecoxib as measured using western blot. Results are presented as % of the control. Each value is a mean ± SD of 3 to 5 determinations. *p<0.05, **p<0.001 (versus control). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0069928), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of MFN1 or MFN2 overexpression on DENV infection.(A) Western blot analysis of A549+on/HA-MFN1 cells induced with Dox (1 μg/ml) or not for 18 h, then infected with DENV serotype 2 for 24 or 48 h by the indicated moi. The relative ratios of band intensity were quantified by ImageJ. p.i. (h): hours post infection. (B) DENV plaque-forming assay of culture supernatants from A549+on/HA-MFN1 cells cultured with or without Dox (1 μg/ml) for 18 h, then infected with DENV (moi 0.1 or 10) as indicated. Data in panels B-D & F-G are mean ± SD (n = 3 per group) & were compared by two-tailed Student’s t test. (C) RT-qPCR analysis of IFN beta mRNA expression at indicated time point in DENV-infected (moi 10) A549+on/HA-MFN1 cells with or without an 18 h-Dox-pretreatment. (D) Analysis of antiviral activity against dSinF-Luc/2A virus in culture media from A549+on/HA-MFN1 cells with DENV infection (moi 5, 48 h) with or without 18-h Dox pretreatment. (E) Western blot analysis of A549+on/HA-MFN2 cells induced with Dox (1 μg/ml, 18 h) or not, then infected with DENV (moi 10) for the indicated time. (F & G) Analysis of the culture supernatants derived from panel E for DENV titer (F) & LDH release (G). (H) Flow cytometry of A549+on/HA-MFN2 cells double stained with JC-1 & annexin V. Cells were infected with DENV (moi 5) for 48 h with or without 18-h Dox pre-treatment. Decreased red/green fluorescence ratio of JC-1 represents disrupted MMP. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1005350), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - HSP90 & HSP70 interact with PABPN1.(A-C) A17-PABPN1 induced INIs in C2C12 cells. Mouse muscle C2C12 myoblasts were transfected with 0.5 μg GFP-tagged A10-PABPN1 or A17-PABPN1 constructs by lipofectamine 2000. Twenty-four hours post-transfection, myoblasts were fixed with 4% PFA, & the nuclei were stained with DAPI (blue). A10-PABPN1 formed small inclusions in nuclear speckles (arrows) whereas A17-PABPN1 formed large INIs (A). Scale bar, 10 μm. Myoblasts were switched to differentiation medium 24 hr after transfection. Fully differentiated myotubes were fixed & stained with DAPI. INIs formed by A10-PABPN1 & A17-PABPN1 are indicated by arrows (B). Cells in 10 random fields with INIs in the nucleus were scored (C). Data are shown as the mean ± SEM, n = 5; *, P < 0.01. (D) Interaction of PABPN1 with HSP90. Lysates from HEK293 cells transfected with Flag-HSP90 & GFP-tagged A17-PABPN1 were immunoprecipitated with an anti-Flag antibody. Immunoprecipitates (IP) were analyzed by Western blot (IB). (E) Interaction of PABPN1 with HSP70. Lysates from HEK293 cells transfected with Flag-HSP70 & GFP-tagged A17-PABPN1 were immunoprecipitated with an anti-Flag antibody. (F) HSP90 associates with the PABPN1 aggregates. C2C12 myoblasts were transfected with A17-PABPN1 constructs, & the cells were processed for immunofluorescence staining using a HSP90 antibody 48 hr after transfection. Arrows indicate the recruitment of HSP90 to the PABPN1 aggregates. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0138936), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Intralipid enhances the biodistribution of paclitaxel in mice organs. (A–E) Determination by LC/MS of biodistribution of paclitaxel in mice organs at 24 or 48 hrs after Abraxane treatment. The organs include liver (A), lung (B), kidney (C), spleen (D), & heart (E). The number above the box denotes the median amount of remaining paclitaxel in mice organs. n.d., not determined. (F) Western blot analysis of the apoptosis signal of the cleaved caspase-3 in the liver at 24 & 48 hrs after treatment with 45 or 90 mg/kg Abraxane with or without Intralipid. The blots of caspase-3 & beta -actin are cropped from different parts of the same gel. (G) The intensities of the apoptosis signals normalized with beta -actin are quantified, & the values summarized in the right box-and-whisker plot, denote the relative intensities compared with those in the control group. (H) Immunohistochemistry of the cleaved caspase-3 in liver sections. Immunohistochemistry was performed on liver tissues in each group, & one case is shown. Original: 200x magnification; Inserts: 400 magnification. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32071352), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on ATP-mediated caspase-1 gene expression & activation in human macrophages.Cells were pretreated with HSM extracts (1 or 2%) for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & subsequently ATP (5 mM) for 1 h. (a) The mRNA expression levels of caspase-1 were determined by RT-PCR analysis. (b) Caspase-1 mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) The secretion of caspase-1 subunit p20 into the supernatants of THP-1 macrophages was assessed by ELISA. (d) Cell lysates & culture supernatants were Western-blotted to detect pro-caspase-1 p45 & caspase-1 subunit p20. Data are presented as means ± SE of three experiments preformed in duplicate. #P < 0.01 versus untreated cells. *P < 0.01 versus HSM-untreated control (ethanol) cells. †P < 0.05 versus HSM (1%) treated cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of androgen & anti-androgen on cell proliferation, cell cycle, & Cdk2 activity in LNCaP 104-R1 cells.(A) Gene expression of CDKN1B was determined in LNCaP 104-R1 cells treated with 0, 0.1, or 10 nM R1881 for 96 h using qRT-PCR. LNCaP 104-R1 cells treated with 10 nm R1881 in the presence or absence of 5 µm anti-androgen Casodex for 96 h. Asterisk * denotes significant difference p<0.05 of the treated cells as compared to control cells. (B) Relative cell number was determined by fluorometric DNA assay. (C) Percentage of cell population in S phase was determined by flow cytometry analysis. Asterisk * denotes a significant difference (p<0.05) of the treated cells as compared to control cells. (D) Histone H1 phosphorylation was assayed using Cdk2 immunoprecipitated from cell lysate containing 2 mg protein. Relative radioactivity was determined by scanning with a Storm 860 phosphoimager (Molecular Dynamics, Sunnyvale, CA, U.S.A.). Protein expression level of Cdk2, p27Kip1, & beta -actin were determined by Western blotting from the same cell lysates. Abundance of beta -actin protein was used as loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25271736), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - ENERGI-F704 attenuates LPS-induced nuclear translocation & production of NF-kappa B in BV2 cells. BV2 cells were stimulated with 200 ng/mL LPS & subsequently treated with ENERGI-F704 incorporating with or without compound C for 1 h. (a) After treatments, cells were fixed for immunocytochemical staining. NF-kappa B & nuclei were visualized using Alexa Fluor 488 (green) & DAPI (blue), respectively. (b) Ratio of nuclear: cytoplasmic immunofluorescence of NF-kappa B was assessed by microscopy image & quantified using ImageJ software. Ratio < 1 indicates brighter cytoplasmic staining for NF-kappa B, whereas ratios > 1 indicate brighter nuclear staining for NF-kappa B. (c) BV2 cells were incubated with 200 ng/mL LPS in the presence of ENERGI-F704 for 24 h. After treatments, cell lysates were used to determine the levels of NF-kappa B using Western blotting. Data are presented as the mean ± SEM of three independent experiments (one-way ANOVA; * & **, P < 0.05 & P < 0.01; specific comparison to LPS-treated control). Representative images of three independent experiments are shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25025067), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - RAF1/ERK signalling is preferentially required for PANC1 cell survival in the absence of exogenous insulin. (A) Effects of different small molecule inhibitors on propidium iodide (PI) incorporation (PI) in PANC1 cells were tracked & expressed as the fold change in the percent of PI & Hoechst co-positive cells over total Hoechst positive cells at that hour relative to t =0 hour. Kinetic data were analyzed relative to serum-free control by two-way ANOVA (n =3) Data points that have been shaded solid black represent statistical significance when compared to non-treated conditions at that time point. # Indicates statistical significance in cells treated with Akti1/2 when compared to control at that time point. (B) Average number of PI positive cells over time of each treated group in Figure 5A is shown as a histogram expressed in arbitrary units (AU). GW5074 exhibited statistical significance, where as other treatments did not yield significance. U0126 p = 0.38, GW5074 *p = 0.0005, Akti-1/2 p = 0.395, Wort. p = 0.292 (n = 3). (C) The effect of 24 hours treatment with inhibitors on cleaved caspase 3 protein levels in PANC1 cells. This is a representative immunoblot of three independent biological replicates (n =3). (D-E) PANC1 cells were serum starved & treated with either DMSO, 10 μM GW5074, 10 μM U0126, 200 nM Akti-1/2 & 1 mM wortmannin (wort.) for 24 hours & 120 hours (n =4-5). Cell viability of PANC1 cells was expressed as the fold change of the treated relative to control. (C-E) One-way ANOVA analysis with Bonferroni post-test was performed. *Represents statistical significance of p < 0.05 where treated groups are compared to control (-) in the post-hoc test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25373319), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Analysis of HCAR expression in D2 & D2G retina & ON. a, b Bar graphs showing Hcar1 (a), ***p = 0.0001, & Hcar2 (b) mRNA normalized to Hprt mRNA by qRT-PCR; n = 8 ON per group. c Immunohistochemical analysis of HCAR1 (red) expression in the untreated (Unt) & keto D2 retina & ON. GFAP (green) labels Müller glia & astrocytes, Iba1 (cyan) labels microglia, & DAPI (blue) stains nuclei. Arrows indicate colocalization of HCAR1 with Iba1+ microglia & GFAP+ astrocytes. d, e Percentage of mean fluorescence of HCAR1 in the inner retina (d), **p = 0.0036, & in the ROI of the proximal ON (e), ***p = 0.0001; five retinae per group, with five sections/retina. f Western blot analysis of HCAR1 protein level in retina. g Quantification by densitometry of HCAR1 protein normalized to beta -actin, ***p = 0.0008; n = 3 blots per group, each with independent samples. h HCAR1 protein in the ON normalized to beta -actin levels, as determined by capillary electrophoresis, **p = 0.0025; n = 3 ON per group. All bar graphs are presented as the mean ± SEM, n = 5, analyzed by two-tailed unpaired t test. Scale bar, 20 μm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30424795), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - iPSC-RPE from an ARB donor show reduced levels of Best1 expression compared to five different iPSC-RPE control lines. (A) To compare Best1 protein expression levels between different iPSC-RPE lines, beta -actin was used as a loading control. (B) While iPSCs displayed no detectable Best1 protein expression, all shown iPSC-RPE lines – regardless of donor origin – displayed detectable Best1 protein expression. (C) The three control iPSC-RPE lines derived from unrelated, unaffected donors all exhibited similar levels of Best1 protein expression. The dad exhibited reduced levels compared to the unrelated controls & the mom showed levels that were about 50% that of the dad. This was expected as the antibody used cannot detect the mutant Best1I366fsX18 which both the mom & the ARB patient are heterozygous for. Best1 expression levels in the ARB iPSC-RPE line were about a quarter of the levels observed in the Mom 214 iPSC-RPE clone. (D) Widefield immunofluorescent staining of Best1 reveals basolateral staining in each of the iPSC-RPE lines, indicating that Best1 protein is properly localized regardless if Best1R141H and/or Best1I366fsX18 are present. Immunofluorescent signal was notably weaker in the ARB iPSC-RPE clones than in the control clones. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-21651-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Nifedipine & the expression p-AMPK, SREBP-1/2(nuclear), PPAR-alpha (nuclear) & lipin-1 (nuclear) in 48 h. (a) The AMPK/actin was upregulated to 107.1%, 112.6%, & 116.4%, significantly at doses of 7.5, 15, & 30 μM (p < 0.05), the ratio p-AMPK/AMPK was downregulated to 82.1%, 58.7%, & 49.9%, respectively. (b,c) SREBP-1 was activated in control. The SREBP-1/HDAC was upregulated to 109.9% & 132.0% by nifedipine at doses of 7.5 & 30 μM, respectively (p < 0.01); The SREBP-2 were elevated to 127.5%, 146.7% & 161.0% by nifedipine at doses of 7.5, 15 & 30 μM, respectively (p < 0.05 in 7.5 μM, p < 0.01 in 15 & 30 μM) (d) nifedipine downregulated PPAR-alpha . The PPAR alpha /Lamin B was suppressed from 80.2%, 85.6%, to 96.4% compared to control (100%) upon administration of nifedipine at doses 7.5, 15 & 30 μM, respectively. (p < 0.05 in 15 & 30 μM) (e) Lipin-1 was activated in control. The lipin-1/lamin B was upregulated after nifedipine treatment to 102.5%, 115.8% & 121.1%, respectively, by nifedipine at doses of 7.5, 15, & 30 μM, respectively (p < 0.05 in 15 & 30 μM). p-values ≤ 0.05 (marked as *) were considered statistically significant. In addition, p-values ≤ 0.01 are marked as **. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30934807), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - The effect of AM1241, SB203580, & PDTC on the thermal threshold & the expression of P2Y12 & P2Y13 receptors. Data are presented as TWL. All values represent mean ± standard deviation. a Comparison of thermal stimulation pain threshold in the ipsilateral hind paw of rats (n = 8). Spinal ADPbetaS injection reduced TWL from days 3 to 7 after ADPbetaS injection. *P < 0.05 compared w/ control group. Pretreated w/ AM1241 (100 PM, 20 min), SB203580 (50 μM), or PDTC (5 μg/10 μl) significantly inhibited the reduced w/drawal threshold days 3 to 7 in ADPbetaS-treated rats, respectively (compared w/ ADPbetaS-treated rats, AM1241: P < 0.05; SB203580: P < 0.05; PDTC: P < 0.05). b RT-PCR results show the expression of P2Y12 mRNA expression in the dorsal spinal cord of the rats in every groups (n = 8 per group). *P < 0.05 means comparison w/ the control group; +P < 0.05 means comparison w/ the ADPbetaS-treated rats. c RT-PCR results show the expression of P2Y13 mRNA expression in the dorsal spinal cord of the rats in every group (n = 8 per group). *P < 0.05 means comparison w/ the control group; +P < 0.05 means comparison w/ the ADPbetaS-treated rats. d Western blotting image of P2Y12R expression. The top panel the target band, P2Y12R, & the bottom one for the loading control beta-actin. e Western blotting quantitative analysis of the P2Y12R expression in the dorsal spinal cord of the rats. *P < 0.05 means comparison w/ the control; +P < 0.05 means comparison w/ the ADPbetaS-treated rats. f Western blotting quantitative analysis of the P2Y13R expression in the dorsal spinal cord of the rats. *P < 0.05 means comparison w/ the control; +P < 0.05 means comparison w/ the ADPbetaS-treated rats Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Intrathecal injection of SB203580 (50 μM) or PDTC (5 μg/10 ml) suppressed the increased expression of P2Y12 & P2Y13R in the dorsal spinal cord of CCI rats. a Comparison of thermal stimulation pain threshold in the ipsilateral hind paw of the rats (n = 8). Data are presented as TWL. CCI reduced TWL from days 1 to 7 after nerve injury. *P < 0.05 compared with control group. Pretreated with SB203580 (50 μM) or PDTC (5 μg/10 ml) significantly inhibited the reduced withdrawal threshold days 1 to 7 in CCI rats, respectively (compared with vehicle-treated CCI group, SB203580: +P < 0.05; PDTC: +P < 0.05). b RT-PCR results show the expression of P2Y12 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the control group; +P < 0.05 means comparison with the vehicle-treated CCI group. c RT-PCR results show the expression of P2Y13 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the control group; +P < 0.05 means comparison with the vehicle-treated CCI group. d Western blotting image of P2Y12R & P2Y13R expression. The top panel was the target band, & the bottom one was for the loading control beta-actin. e Western blotting quantitative analysis of the P2Y12R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group. f Western blotting quantitative analysis of the P2Y13R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Effects of HSM on the expression of P2X4R & P2X7R & ROS production in human macrophages.Cells were pretreated with various concentrations (1 or 2%) of HSM extract for 20 h, followed by treatment with LPS (0.5 μg/ml) for 3 h & ATP (5 mM) for 1 h. (a) The mRNA expression levels of P2X4R & P2X7R were determined by RT-PCR, using beta -actin as the internal control. (b) P2X4R & P2X7R mRNAs were quantified using real-time PCR. beta -actin gene expression was used for normalization. The results are expressed as fold changes, considering one as the value of untreated cells. (c) Cell lysates were analyzed by Western blot analysis used specific anti-P2X4R & anti-P2X7R antibodies. (d) ROS production was measured with the total ROS detection kit, using a fluorescence microplate reader. Pyocyanin (200 μM), a ROS inducer, was used as a positive control for ROS formation. Data are presented as means ± SE of three experiments preformed in duplicate. #P < 0.01 versus untreated cells. versus HSM-untreated control (ethanol) cells. *P < 0.01 versus HSM-untreated control (ethanol) cells. †P < 0.05 versus HSM (1%) treated cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23459183), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Intrathecal injection of AM1241 (100pM) attenuated CCI-induced hyperalgesia & increased expression of p-p38MAPK & NF-kappaBp65 in the rat dorsal spinal cord (n = 8 per group). a Western blotting image of p-p38MAPK expression. The top panel was the target band, p-p38MAPK, & the bottom one was for the loading control beta-actin. b Western blotting quantitative analysis of the p-p38MAPK expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group. c Western blotting image of NF-kappaBp65 expression. The top panel was the target band, NF-kappaBp65, & the bottom one was for the loading control beta-actin. d Western blotting quantitative analysis of the NF-kappaBp65 expression in the dorsal spinal cord of the rats. *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the vehicle-treated CCI group Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - Intralipid enhances the cytotoxicity of Abraxane in tumors. (A) The box-and-whisker plot represents the amount of paclitaxel in tumors determined by LC/MS. The number above the box denotes the median amount of remaining paclitaxel in the tumor tissues. (B) Western blot analysis of the apoptosis signal, cleaved caspase-3, in the tumors at 24 & 48 hrs after Abraxane treatment. beta -actin served as a loading control. The blots of caspase-3 & beta -actin are cropped from different parts of the same gel. The intensity of each band of the cleaved caspase-3 was measured by ImageJ software (NIH) & normalized to its own signal of beta -actin. The values summarized in the box-and-whisker plot denote the relative intensities compared with those in the control group. (C,D) The immunohistochemistry of cleaved caspase-3 (C) & the TUNEL assay (D) are analyzed in 48 hrs tumor sections. Original: 200x magnification; Inserts; 400x magnification. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32071352), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - The effect of minocycline on the expression of P2Y12 & P2Y13 receptors. All values represent mean ± standard deviation. a RT-PCR results show the expression of P2Y12 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the CCI rats. b RT-PCR results show the expression of P2Y13 mRNA expression in the rat dorsal spinal cord (n = 8 per group). *P < 0.05 means comparison with the sham group; +P < 0.05 means comparison with the CCI rats. c Western blotting quantitative analysis of the P2Y12R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the control; +P < 0.05 means comparison with the ADPbetaS-treated rats. d Western blotting quantitative analysis of the P2Y13R expression in the rat dorsal spinal cord. *P < 0.05 means comparison with the control; +P < 0.05 means comparison with the ADPbetaS-treated rats. e Western blotting image of P2Y12R expression. The top panel was the target band, P2Y12R, & the bottom one was for the loading control beta-actin. f Western blotting image of P2Y13R expression. The top panel was the target band, P2Y13R, & the bottom one was for the loading control beta-actin Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-017-0960-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - ENERGI-F704 decreases LPS-induced NO, iNOS, COX-2, & PGE2 production in BV2 cells. BV2 cells were stimulated with 200 ng/mL LPS & subsequently treated with either ENERGI-F704 or AICAR for 24 h. (a) Levels of NO in the culture of BV2 cell were determined. (b) After treatments, cell lysates were used to determine the level of iNOS & COX-2 using Western blotting. (c) LPS-treated BV2 cells were treated with ENERGI-F704 in cotreatment with or without compound C for 24 h. Cell lysates were used to determine the level of iNOS & COX-2 using Western blotting. (d) LPS-induced BV2 cells were treated with ENERGI-F704 in cotreatment with or without compound C for 48 h, & the level of PGE2 in the culture medium was determined. Data are presented as the mean ± SEM of three independent experiments (two-way ANOVA; *, **, & ***, P < 0.05, P < 0.01, & P < 0.01; specific comparison to LPS-treated control). Representative images of three independent experiments are shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25025067), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Simple Western: beta-Actin Antibody (AC-15) [NB600-501] - iPSC-RPE from all donors express RPE marker proteins. (A) As a loading control, each iPSC-RPE line (ARB patient, the patient’s parents, & three unrelated, unaffected controls) as well as iPSCs (negative control) were blotted for beta -actin. (B) Unlike iPSCs, all iPSC-RPE lines showed robust expression of CRALBP. (C) Just like CRALBP, iPSC-RPE from the patient, the patient’s mom, the patient’s dad, & the three unrelated, unaffected controls all expressed RPE65. The negative control (iPSCs) had no detectable RPE65 protein expression. (D) Each of the iPSC-RPE lines but not iPSCs displayed strong expression of the RPE marker Mer-tk. (E) The relative expression levels for each of these three markers is summarized for iPSCs & all of the iPSC-RPE lines. Graphs show the summary of expression for each marker from three separate blotting experiments. Variability between each of the controls as well as between the different iPSC-RPE lines was observed for each of the RPE markers. Control 4 displayed the highest levels of CRALBP, Control 138 had the highest levels of RPE65, & Control 1 showed the highest levels of Mer-tk. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-21651-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: Mouse Monoclonal beta-Actin Antibody (AC-15) [NB600-501] - Quantification of TNF alpha in bone graft in the tibia of intrailiac cellularized goats at different times compared with tibial autograft and untreated bone defect. Beta-actin antibody (Catalog # NB600-501) was used at a dilution of 1:3000 and TNF alpha antibody (Catalog # NBP1-97453) was used at a dilution of 1:1000. Evaluation of pre-cellularized graft in iliac crest in the treatment of bone defects in the tibia of goats. G1: cellularized graft for 4 weeks applied to tibial bone defect, G2: cellularized graft for 6 weeks applied to tibial bone defect, G3: cellularized graft for 8 weeks applied to tibial bone defect, G4: tibial autograft and G5: untreated bone defect. All groups were evaluated 10 days after graft implantation. Image from a verified customer review.

Reactivity:

Hu, Mu, Rt, Po, Bv, Ca, Ch, Fe, Fi, Gp, Gt, Ha, Le, Ma, Pm, Rb, Sh, Sq, Tr, Ze, Dr(-)

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Bv, Ca, Ch, Fe, Fi, Gp, Gt, Ha, Le, Ma, Pm, Rb, Sh, Sq, Tr, Ze, Dr(-)

Applications:

, , , , , , , , , , ,

Applications: , , , , , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB110-67828AF350
Alexa Fluor 405	NB110-67828AF405
Alexa Fluor 488	NB110-67828AF488
Alexa Fluor 532	NB110-67828AF532
Alexa Fluor 594	NB110-67828AF594
Alexa Fluor 647	NB110-67828AF647
Alexa Fluor 700	NB110-67828AF700
Alexa Fluor 750	NB110-67828AF750
Alexa Fluor Plus 488	NB110-67828AFP488
Alexa Fluor Plus 555	NB110-67828AFP555
Alexa Fluor Plus 594	NB110-67828AFP594
Alexa Fluor Plus 647	NB110-67828AFP647
Alexa Fluor Plus 680	NB110-67828AFP680
Biotin	NB110-67828B
CoraFluor 1	NB110-67828CL1
DyLight 350	NB110-67828UV
DyLight 405	NB110-67828V
DyLight 488	NB110-67828G
DyLight 550	NB110-67828R
DyLight 594	NB110-67828DL594
DyLight 650	NB110-67828C
DyLight 680	NB110-67828FR
DyLight 755	NB110-67828IR
FITC	NB110-67828F
HRP	NB110-67828H
Janelia Fluor 525	NB110-67828JF525
Janelia Fluor 549	NB110-67828JF549
Janelia Fluor 646	NB110-67828JF646
Janelia Fluor 669	NB110-67828JF669
PerCP	NB110-67828PCP
mFluor Violet 450 SE	NB110-67828MFV450
mFluor Violet 500 SE	NB110-67828MFV500
mFluor Violet 610 SE	NB110-67828MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB110-67828

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AF-401-NA

IL-1 beta/IL-1F2 Antibody [Unconjugated]

(3 Reviews)

(610 Publications)

100 μg

AF-401-NA

HAF017). A specific band was detected for IL-1 beta /IL-1F2 at approximately 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_lightbox">

<b>Biological Strategies Validation. </b>Western blot shows lysates of THP-1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200 nM PMA for 24 hours and 10 µg/mL LPS for 4 hours and RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours. PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (<a class=

HAF017). A specific band was detected for IL-1 beta /IL-1F2 at approximately 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." alt="Western blot shows lysates of THP-1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200 nM PMA for 24 hours and 10 µg/mL LPS for 4 hours and RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours. PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for IL-1 beta /IL-1F2 at approximately 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_thumb" />

NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_lightbox">

<b>Biological Strategies Validation. </b>IL-1 beta /IL-1F2 was detected in immersion fixed MCF-7 human breast cancer cell line using Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; <a class=

NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_thumb" />

CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections." class="big_lightbox">

IL-1 beta /IL-1F2 was detected in perfusion fixed frozen sections of mouse thymus using Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; <a class=

CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections." class="big_thumb" />

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_lightbox">

<b>Biological Strategies Validation. </b>Simple Western lane view shows lysates of RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 24 hours, loaded at 0.5 mg/mL. A specific band was detected for IL-1 beta /IL-1F2 at approximately 40 kDa (as indicated) using 2.5 µg/mL of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (<a class=

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_thumb" />

401-ML) stimulates proliferation in the the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line) as measured by Resazurin (AR002). Proliferation elicited by Recombinant Mouse IL-1 beta /IL-1F2 (50 pg/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA). The ND₅₀ is typically ≤ 0.25 µg/mL." class="big_lightbox">

Recombinant Mouse IL-1 beta /IL-1F2 (<a class=

401-ML) stimulates proliferation in the the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line) as measured by Resazurin (AR002). Proliferation elicited by Recombinant Mouse IL-1 beta /IL-1F2 (50 pg/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Mouse IL-1 beta /IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA). The ND₅₀ is typically ≤ 0.25 µg/mL." class="big_thumb" />

VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in epithelial cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_lightbox">

IL‑1 beta /IL‑1F2 was detected in perfusion fixed paraffin-embedded sections of Mouse Intestine using Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Sheep IgG VisUCyte™ HRP Polymer Antibody (<a class=

VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in epithelial cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_thumb" />

<b>Biological Strategies Validation. </b>Quercetin inhibits auto-reactive NLRP3 inflammasome.(A) IL-1 beta concentrations in Nlrp3A350V/A350V and WT BMDM culture supernatants primed with LPS (500 ng/ml) for 90 min and treated with quercetin 1 h after LPS. (B) Supernatants of BMDM were analyzed by anti-ASC, anti-Caspase-1 immunoblotting. Lysates of BMDM were analyzed by anti-IL-1 beta and anti-beta -actin immunoblotting. Data shown are representative of two or more experiments (means ± SD) ***p < 0.001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28148962), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>GRA7-I-induced inflammasome activation was required for antimicrobial activity in MTB-infected macrophages.(A and B) BMDMs from PKC alpha +/+ and PKC alpha -/- mice (A) and BMDMs were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-ASC for 2 days (B) infected with MTB (MOI = 1) for 4 h and then stimulated with rGRA7 (1, 5, 10 μg/ml) and its mutants for 18 h. IB analysis for IL-1 beta p17, IL-18 p18, or caspase-1 p10 in supernatants (SN), ASC, NLRP3, pro-IL-1 beta , pro-IL-18, or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. (C and D) Intracellular survival of MTB was assessed by CFU assay. BMDMs were infected with MTB for 4 h, followed by treatment with rGRA7, and then lysed to determine intracellular bacterial loads. The data are representative of five independent experiments with similar results (A and B). Data shown are the mean ± SD of five experiments (C and D). Significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) compared with rVector. CFU, colony-forming units. ns, not significant. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>M. bovis-induced inflammasome activation requires ERS. (A) Immunoblot analysis at different time-points of Bip and p-IRE1 alpha in lysates of BMDMs infected with M. bovis (MOI 10). (B) Immunoblot analysis at different time-points of IL-1 beta in supernatants of BMDMs infected with M. bovis (MOI 10). (C) ELISA for IL-1 beta analysis from supernatants of BMDMs infected with M. bovis (MOI 10). (D) IL-1 beta immunoblot analysis of supernatants from BMDM infected for 24 h with M. bovis (MOI 10) in the presence or absence of 4-PBA. (E) ELISA for IL-1 beta quantification from supernatants of BMDMs treated with or without 4-PBA for 1 h and then infected with M. bovis (MOI 10) for 24 h. ELISA for (F) TNF- alpha and (G) IL-6 detection in supernatants from M. bovis (MOI 10) infected BMDMs. ELISA for (H) TNF-alpha and (I) IL-6 detection from supernatants of BMDMs treated with or without 4-PBA for 1 h and then infected with M. bovis (MOI 10) for 24 h. TM, tunicamycin, positive control for ERS induced mitochondrial damage, 10 μg/mL; LPS+ATP, positive control for inflammasome activation, 200 ng/mL, and 1 mM, respectively; LPS, positive control for TNF-alpha and IL-6 production, 200 ng/mL; UNT, untreated; 4-PBA, 4-phenyl butyric acid, ERS inhibitor, 5 mM; MOI, multiplicity of infection. For (C,F,G), Data are representative of at least three independent experiments, each performed in triplicate for WB. The results are shown are the mean ± SD. The asterisks indicate statistically significant differences compared with untreated cells (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). P-values were obtained by using one-way ANOVA followed by post-hoc Turkey' comparison test. For (E,H,I), data are representative of at least three independent experiments. The results are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. P-values were analyzed using Student's t-test. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30846986)

<b>Biological Strategies Validation. </b>NLRP1A is activated by N-terminal proteolysis.(A) The amino acid sequence of the first 244 residues of NLRP1AB6 was aligned to NLRP1B129 and NLRP1BB6. The arrow above the alignment indicates the LF-cleavage site in NLRP1B129. (B) 293T cells were transfected for 36h with 200ng empty vector (V) or plasmids pcDNA3.1-HA-NLRP1B129 or -NLRP1AB6-MYC (CMV promoter) along with 200ng mCASP1 and 200ng mIL-1 beta expression vectors. Cells were treated overnight with anthrax lethal toxin (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. (C) 293T cells were transfected for 36h with plasmids encoding 400ng GFP-HA-NLRP1B129 or GFP-HA-NLRP1AB6 (under the control of the LTR promoter in pMSCV) or with mutants engineered to contain an N-terminal TEV protease site. Cells were also co-transfected with 100ng empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), plus 300ng empty pMSCV. (D) To assess IL-1 beta cleavage, cells were transfected as in C, but with 8ng of GFP-HA-NLRP1B and co-transfected with vectors encoding 200ng mCASP1 and 200ng mIL-1 beta . For panels B-D, data shown are representative of at least three similar experiments. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.

Colocalization of phagosome markers with internalized Bb in WT and MyD88−/− BMDMs. a-b Confocal 40x images of WT (a) and MyD88−/− (b) BMDMs after 6 h stimulation with Bb at MOI 10:1, depicting colocalization of Bb-containing phagosomes with LAMP-1. White box indicates phagosome depicted in inset. Graph shows the intensity of each indicated pixel across the white line (distance on x-axis). Green is Bb, red is LAMP-1 and yellow is actin. c Quantitation of colocalization between Bb and LAMP-1 in 10 phagosomes of WT (black dots) and MyD88−/− (red dots) BMDMs by measuring intensity difference between LAMP-1 staining and Bb staining. d Western blot of protein lysate isolated from WT BMDMs after 6 h stimulation with Bb +/− ATP (C = cell lysate, S = supernatant) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34000990), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>Differential effects of IL-1 beta neutralization on melanoma lung nodules in WT and DJ-1 KO mice.(A) IL-1 beta antibody (10μg) or control IgG was intravenously injected into mice once. The melanoma cells (6×104) were intravenously injected 30 minutes later. The mice were sacrificed three weeks later. Bar chart showed that the anti-IL-1 beta neutralizing antibody can effectively lower serum IL-1 beta levels as compared with control IgG in both WT and DJ-1 KO mice. Note that serum IL-1 beta levels were higher in DJ-1 KO mice than those in WT mice. (B) Inhibition of IL-1 beta neutralization on lung nodules formation in DJ-1 KO mice. Upper panels: melanoma nodules in lungs of WT (top) and KO (bottom) mice treated with control IgG (left) or neutralizing anti-IL-1 beta antibody (right). For each experimental condition, the gross and microscopic morphologies of tumor nodules are indicated by arrows, and are shown at left and right sites, respectively. Scale bars: 0.5 mm for lung photographs and 0.5 mm for H&E staining. Lower panel: Bar chart showed the summarized results of lung nodule numbers in WT and DJ-1 KO mice. Note that IL-1 beta neutralization enhanced and suppressed melanoma nodules formation in WT and DJ-1 KO mice, respectively. Data are presented as mean ± S.E.M. n = 5 for each group, * p < 0.05 compared with control IgG-treated WT mice, # p<0.05 compared with respective control IgG in WT and DJ-1 KO mice. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pro-IL-1 beta degradation is induced by the autophagy inhibitors chloroquine and bafilomycin A, and by the proteasome activator betulinic acid. (A) Chloroquine selectively attenuates IL-1 beta release from cardiac fibroblasts. The cells were incubated with LPS and/or 20 μM chloroquine phosphate as indicated for 18 h, then activated with 3 mM ATP for 60 min. IL-1 beta , TNF, MIP-2, and IL-6 were quantified in the conditioned media (n = 3). (B,C) Cardiac fibroblasts were incubated with LPS for 18 h with or without 20 μM chloroquine phosphate and pro-IL-1 beta quantified with western blot (n = 3). (D) Pro-IL-1 beta mRNA expression was quantified with qPCR (n = 3). (E,F) Cardiac fibroblasts were incubated with LPS for 20 h with or without 10% FCS or 20 μM chloroquine phosphate as indicated. The amount of total ubiquitinated protein was quantified with western blot (n = 4). (G,H) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 100 nM bafilomycin A1 for 20 h. Pro-IL-1 beta was quantified with western blot. (I,J) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 20 μM betulinic acid as indicated for 20 h. Pro-IL-1 beta was quantified with western blot. All columns are mean with SEM. *p < 0.05, **p < 0.01 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>The B6 variant of NLRP1B is not cleaved by LF but can form an inflammasome in response to proteolysis.(A) The amino acid sequence of the first 244 residues of NLRP1BB6 was aligned to the homologous sequence of NLRP1B129. The arrow above the alignment indicates the LF-cleavage site in NLRP1B129. Asterisks indicate sites of amino acid identity. (B) For detection of NLRP1B expression, 293T cells were transfected with the indicated amounts of and empty vector (V) or plasmids encoding GFP-HA-NLRP1B129 or GFP-HA-NLRP1BB6 (construct schematics and the predicted molecular weight of each protein is depicted in the upper panel). Cells were treated overnight with anthrax lethal toxin protein (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. For NLRP1 expression and cleavage, CASP1 and IL1B were omitted to prevent cell death and resulting apparent differences of expression. For blots probed with anti-HA (NLRP1B), the lysates were not boiled prior to loading to prevent aggregation and smearing of full-length and FIIND-processed NLRP1B on the immunoblot. To visualize the N-terminal processed form of NLRP1B, the lysates were boiled and resolved on a separate gel. (C) 293T Cells were transfected with the same amount of titrated NLRP1B encoding plasmid and treated as in B, but transfections also included plasmids encoding mouse CASP1 (200ng), IL-1 beta (200ng) and 200ng of empty vector. (D) For detection of NLRP1B expression, 293T cells were transfected for 36h with 250ng of plasmids encoding WT 129 or B6 NLRP1B or mutants engineered to express the TEV-protease site. Each plasmid was co-transfected with either 100ng of empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), supplemented with 300ng empty vector (pMSCV). Cells were not treated with LeTx and lysates were analyzed by immunoblotting as in (B). (E) Cells were treated as in C, but with 8ng of plasmids encoding NLRP1B, al

Rapamycin increases pro-IL-1 beta protein levels, rescues serum starvation-induced pro-IL-1 beta degradation, inhibits mTOR but induces no autophagy in LPS-stimulated cardiac fibroblasts. (A) IL-1 beta release was quantified in conditioned media from cardiac fibroblasts primed with 10 ng/mL LPS for 20 h with or without 500 nM rapamycin, as indicated, prior to activation with 3 mM ATP for 60 min. (B,C) Fibroblasts were primed with 10 ng/mL LPS for 20 h with or without 500 nM rapamycin and pro-IL-1 beta western blot performed (n = 3). (D,E) Control cells were incubated with 10% FCS, while serum deprived cells were incubated with or without 500 nM Rapamycin. Protein expressions were quantified with western blot (n = 4) (F,G). IL-1 beta and TNF-alpha were quantified in conditioned media from cardiac fibroblasts incubated with or without 10% FCS and 500 nM rapamycin and stimulated with LPS 10 ng/mL for 20 h prior to activation with 3 mM ATP for 60 min as indicated (n = 6) (H,I) IL-1 beta and TNF mRNA were quantified with PCR. (J–L) Cardiac fibroblasts were incubated with 10% FCS with or without 10 ng/mL LPS and/or 500 nM rapamycin for 20 h and mTOR (K) and p70 S6 kinase (L) activity were quantified with western blot (n = 5). (M,N) Cardiac fibroblasts primed with LPS (10 ng/mL) were incubated with 10% FCS (control) or no serum for 4 h. Cells were labeled with anti-LC3B and Hoechst and whole slides scanned with an automated immunofluorescence microscope. LC3B puncta in all cells in were automatically counted and ratio to number of Hoechst-labeled kernels calculated. Columns are mean with SEM of paired data normalized to control = 1 (n = 5). (O) Cardiac fibroblasts were incubated with 10% FCS, 10 ng/mL LPS, and/or 5 mM 3-methyladenine (3-MA) for 18 h and/or ATP 3 mM for 60 min as indicated (n = 3). Rapa: rapamycin. 3-MA: 3-methyladenine, ns: not significant, *p < 0.05, **p < 0.01, ****p < 0.0001 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubm

SHP deficiency increases caspase-1 cleavage and IL-1 beta maturation.(a–e) LPS-primed BMDMs from SHP+/+ and SHP−/− mice were stimulated with ATP (5 mM, a,d), nigericin (15 μM, b,e), or transfected with polyinosine–polycytidylic acid (poly I:C, 5 μg ml−1, c) for the indicated durations. (a–c) Supernatants were collected and subjected to ELISA for interleukin (IL)-1 beta , IL-18 and tumour necrosis factor (TNF)-alpha . (d,e) IB analysis for IL-1 beta p17 or caspase-1 p10 in supernatants (SN), SHP, pro-IL-1 beta or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. *P<0.05; **P<0.01; ***P<0.001, compared with SHP+/+ cell cultures (two-tailed Student’s t-test). Data are the means±s.d. of values from four independent experiments (a–c). Data are representative of three independent experiments with similar results (d,e). Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>ERS mediates M. bovis-induced inflammasome activation in vivo. (A) ELISA detection of IL-1 beta in serum samples from C57BL/6 mice treated with or without 4-PBA (18.6 mg/mouse/day) and infected with M. bovis (CFU 200) (n = 7). (B) Immunoblot analysis of NLRP3, AIM2, pro-IL-1 beta , and IL-1 beta in the lung tissues of M. bovis-infected mice in the presence or absence of 4-PBA. (C,D) Pathological lesions (H&E staining) in the lung of mice infected with M. bovis for 3 weeks (C) or 6 weeks (D) in the presence or absence of 4-PBA (18.6 mg/mouse/day) (n = 3). 4-PBA, 4-phenyl butyric acid, ERS inhibitor, 18.6 mg/mouse/day; CFU, colony forming units (n = 3). The results are shown as the mean ± SD. ***P < 0.001, n.s., not significant. P-values were analyzed by using Student's t-test (C,D). Scale bar = 100 μm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30846986), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>POH1 negatively modifies K63-linked polyubiquitination of pro-IL-1 beta . a BMDMs stimulated with LPS for the indicated time periods were immunoprecipitated (IP) with an anti-POH1 or control IgG antibody and immunoblotted (IB) with the indicated antibodies. b HEK293T cells transfected with His-POH1 and either Flag-NLRP3, Flag-caspase-1 or Flag-ASC were subjected to IP with an anti-Flag antibody and IB with an anti-His antibody. c The lysates of HEK293T cells expressing Flag-POH1 and His-pro-IL-1 beta were IP with an anti-Flag antibody and IB with an anti-His antibody in cell lysates. d HEK293T cells transfected with Vsv-ASC, V5-tagged pro-caspase-1, HA-tagged K48-only or K63-only Ub and His-tagged pro-IL-1 beta , along with or without Flag-tagged POH1, were subjected to IP with an anti-V5 antibody or anti-His antibody and then IB with the indicated antibodies. ePoh1 delta /+ and Poh1 delta / delta BMDMs treated as indicated were subjected to IP with an anti-pro-IL-1 beta antibody and IB with the indicated antibodies. f HEK293T cells were transfected with Flag-caspase-1, Vsv-ASC, His-pro-IL-1 beta , Flag-POH1 (WT) or Flag-H113Q-POH1, along with HA-tagged K63-only Ub, then cells were subjected to IP with an anti-His or control IgG antibody and IB with the indicated antibodies. Similar results were obtained from three independent experiments Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>BTK inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1 beta in supernatants and immunoblot analysis of human IL-1 beta p17, caspase-1 p20/p22 in supernatants and pro-IL-1 beta in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1 beta (b,c) and TNF-alpha (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1 beta and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1 beta in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1 beta and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1 beta p17, caspase-1 p20 in supernatants, pro-IL-1 beta , pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1 beta in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26059659), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>SHP inhibits the signal 2 activation of NLRP3 inflammasome.(a,b) LPS-primed BMDMs were treated with fenofibrate (10, 20, 50 μM for 4 h), or (c,d) LPS-primed THP-1 cells were treated with recombinant macrophage-stimulating protein (MSP 10, 50, 100 ng ml−1 for 4 h), and then activated with ATP (5 mM) for 30 min, followed by IB analysis of IL-1 beta p17 or caspase-1 p10 in supernatants (SN), pro-IL-1 beta or pro-caspase-1 in whole-cell lysates (WCL), with actin as a loading control. Data are representative of three independent experiments (a,c). Supernatants were collected and subjected to ELISA for IL-1 beta , IL-18, TNF-alpha and IL-8. Data are the means±s.d. of values from four independent experiments (b,d). *P<0.05; **P<0.01; ***P<0.001, compared with control (two-tailed Student’s t-test). FF, fenofibrate. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>Effects of IL-1 beta neutralization on the accumulation of MDSCs in lungs.(A) Immunofluorescent photos showed that Gr-1+ (green)/CD11b+ (red) cells indicate MDSCs and increased in lungs of DJ-1 KO mice. Treatment with anti-IL-1 beta neutralizing antibody antagonized the accumulation of MDSCs. DAPI was used to stain nuclei. (B) Bar chart showed the summarized results of MDSCs in lungs of WT and DJ-1 KO mice. Note that the neutralizing antibody significantly reduced the number of MDSCs accumulated in lungs. n = 9 for each group, * p < 0.05 compared with control IgG-treated WT mice, # p<0.05 compared with respective control IgG in WT and DJ-1 KO mice. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pro-IL-1 beta maturation is regulated by POH1-mediated deubiquitination. a, b HEK293T cells were transfected with plasmids encoding Flag-caspase-1, Vsv-ASC, His-pro-IL-1 beta (WT) or His-pro-IL-1 beta -K133R with different amounts (125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, the secretion of a IL-1 beta in the supernatants was quantified by ELISA 36 h after transfection; b the cell lysates were IB with the indicated antibodies. c Mutants of murine pro-IL-1 beta with replacement of various lysine residues. d-f HEK293T cells were transfected with Flag-POH1, V5-caspase-1, V5-ASC, His-pro-IL-1 beta (WT) or its mutants, along d with or e-f without HA-tagged K63-only Ub, then d cells were subjected to IP with an anti-His antibody and IB with the indicated antibodies. e The IL-1 beta levels in supernatants were measured by ELISA; or f the cell lysates were collected and IB with the indicated antibodies. g Proposed function of POH1 as a negative regulator in mediating IL-1 beta processing. POH1, upregulated by TLR3/4 activation, interacts with and deubiquitinates pro-IL-1 beta , resulting in restriction of IL-1 beta cleavage and secretion. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey’s post-hoc test) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

Serum starvation selectively induces pro-IL-1 beta degradation in cardiac fibroblasts. (A) Cells were incubated with 10 ng/mL LPS for 5 or 20 h with or without 10% FCS and western blot analysis of pro-IL-1 beta and NLRP3 performed. Blots are representative of 3 independent experiments. (B) Protein bands were quantified and the ratios to beta -actin normalized to mean control = 1 (n = 3). (C,D) Cardiac fibroblasts were incubated with or without 10% FCS and primed with LPS for 20 h. Western blot analysis of complex II (mitochondrial mass marker) and NLRP3- inflammasome protein components (NEK7, NLRP3, ASC, pro-caspase-1, and pro-IL-1 beta ) were performed. Bands were quantified and the ratios to beta -actin normalized to mean control = 1 (n = 4, for ASC n = 10). (E,F) Cardiac fibroblasts (120,000 cells per well seeded in 6 well plates) were incubated with 10 ng/mL LPS for 20 h, with or without 10% FCS, then stained with 500 nM MitoTracker Deep Red for 45 min before MitoTracker fluorescence intensity was quantified with flow cytometry analysis (633 nm laser). All cells were analyzed and all gated cells included in the analysis. The presented dot plots (E) show the gate in red and are representative for 9 biological repeats. Median MitoTracker intensity with and without 10% FCS are shown (F). All columns are mean with SEM. *p < 0.05,**p < 0.01 (paired student t-test). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.

Inflammasome assembly and mature IL-1 beta release in the spinal cord after sciatic nerve axotomy. a Speck-like costaining of NLRP3 and ASC in injured neurons 3 days after the injury (arrows). b Representative WB images of mature IL-1 beta and beta -actin proteins 3 days after unilateral axotomy of sciatic nerve. c Quantification of active IL-1 beta protein expression. N = 3 animals/group. The graph shows values normalized to beta -actin levels and relative to contralateral side. Mean values are shown on bars. Bars represent average ± SEM. **p < 0.01 (ANOVA with Bonferroni post hoc, ax. + vehicle vs. contralateral side). #p < 0.05, ##p < 0.01 (ANOVA with Bonferroni post hoc, compared to ax. + vehicle). Ax. axotomy Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35305649), licensed under a CC-BY license. Not internally tested by R&D Systems.

$GRA7-I activated inflammasomes in an ASC-binding dependent manner.(A) Bacterially purified 6xHis-GRA7-I and its mutants were analyzed by Coomassie blue staining (left) or IB with alpha His (right). (B) BMDMs from PKC alpha +/+ and PKC alpha -/- (left) or BMDMs were transduced with lentivirus-shRNA-NS or lentivirus-shRNA-ASC (MOI = 100) with polybrene (8 μg/mL) (right) for 2 days, the cells were stimulated with rGRA7 (5 μg/ml) and its mutants for the indicated times and culture supernatants were harvested and analyzed for cytokine ELISA for IL-1 beta and IL-18. Data shown are the means ± SD of five experiments. Significant differences (*P < 0.05; ***P < 0.001) compared with PKC alpha +/+ or shRNA-NS. (C) IB analysis for IL-1 beta p17, IL-18 p18, or caspase-1 p10 in supernatants (SN), ASC, NLRP3, pro-IL-1 beta , pro-IL-18, or pro-caspase-1 in whole-cell lysates (WCL). Actin was used as a loading control. (D) IB analysis of lysates of BMDMs as in B and C solubilized with Triton X-100–containing buffer, followed by cross-linkage of insoluble fractions with disuccinimidyl suberate to capture ASC oligomers and analysis of those fractions (I + DSS) and soluble fractions (S) with antibody to ASC. Actin was used as a loading control. (E) Fluorescence confocal images showing formation of speck-like ASC pyroptosomes in BMDMs from PKC alpha +/+ and PKC alpha -/- mice were stimulated with rGRA7 and its mutants for 18 h, fixed, immunostained with antibodies for ASC (Alexa 488). Scale bar, 10 μm. The data are representative of five independent experiments with similar results (A and C-E). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.$

<b>Biological Strategies Validation. </b>POH1 inhibits inflammasome-induced pro-IL-1 beta processing. a-c BMDMs from Poh1 delta /+ and Poh1 delta / delta mice were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a IL-1 beta , b IL-6 and c TNF alpha levels in supernatants were analysed by ELISA. d-f BMDMs lentivirally transduced with control (Vector) or POH1 were stimulated as in a, d IL-1 beta , e IL-6 and f TNF alpha levels in supernatants were analysed by ELISA. g, h BMDMs g derived from Poh1 delta /+ and Poh1 delta / delta mice or h infected by indicated lentiviruses were treated as in a, the cell lysates (CL) and supernatants (SN) were collected and immunoblotted with the indicated antibodies. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. **p < 0.01, ***p < 0.001 (two-tailed Student’s t-test) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>Quercetin inhibits NLRP3 and AIM2 Inflammasomes.LPS (500 ng/ml)-primed BMDM were treated with the indicated flavonoids and concentrations or vehicle (DMSO 0.01%), then stimulated with (A) 5 mM ATP (B) 10 μM Nigericin (C) 130 μg/ml Alum or (D) 400 ng/ml Poly (dA:dT) 30 minutes after treatment. (A–D) IL-1 beta and (E) TNF-alpha concentration in the culture supernatant were measured by ELISA. (B,F,G) LPS-primed BMDM were stimulated with Nigericin treated with quercetin 30 minutes before secondary stimulation. Supernatants and Lysate of BMDM were analyzed by immunoblotting. Cells were stained with FLICA and Caspase-1 activation was determined by FLICA positivity. Data shown are representative of two or more independent experiments (means ± SD) *p < 0.05, **p < 0.01, ***p < 0.001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28148962), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>P. gingivalis and its OMVs differentially induce inflammasome signaling and pyroptosis in human macrophages. MDM were infected as before (2 h at MOI of 25:1, see Materials and Methods) with viable P. gingivalis (Pg), heat-killed-Pg (HK-Pg), OMVs, or heat-inactivated-OMVs (HI-OMVs) and (A) the activation of inflammasome components in the lysates was measured after 24 h by Western blot; beta -actin serves as a loading control throughout. Western blot data are representative of at least three independent experiments. (B) Production of IL-1 beta and IL-18 (by ELISA) from MDM was measured in the supernatant at 24 h. Data are mean ± SD from four independent experiments. Cell viability was determined by measuring extracellular LDH release and by 7-AAD exclusion (by flow-cytometry) at 24 h. 7-AAD is a membrane impermeant dye that is excluded from viable cells. The percent of 7-AAD positive (±SD) cells from three independent experiments was calculated relative to the untreated control. MDM treated with H2O2 (1 mM for 60 min) were included as positive control for 7-AAD. A representative histogram is shown. Cells were also treated where indicated with OMVs in the presence of KYT-1 and KYT-36 (10 μM of each). *p < 0.05: n.s, not significant. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>P. gingivalis and its OMVs differentially induce inflammasome signaling and pyroptosis in murine macrophages. BMM were infected as before (2 h at MOI of 25:1, see Materials and Methods) with viable P. gingivalis (Pg), heat-killed-Pg (HK-Pg), OMVs, or heat-inactivated-OMVs (HI-OMVs) and (A) the activation of inflammasome components in the lysates [or supernatants (sup) where indicated] measured after 24 h by Western blot; beta -actin serves as a loading control throughout. Western blot data are representative of at least three independent experiments. (B) Production of IL-1 beta and IL-18 (by ELISA) from BMM was measured in the supernatant at 24 h. Data are mean ± SD from four independent experiments. Cell viability was determined by measuring extracellular LDH release and by 7-AAD exclusion (by flow-cytometry) at 24 h. 7-AAD is a membrane impermeant dye that is excluded from viable cells. The percent of 7-AAD positive (±SD) cells from three independent experiments was calculated relative to the untreated control. BMM treated with H2O2 (1 mM for 60 min) were included as positive control for 7-AAD. A representative histogram is shown. Cells were also treated where indicated with OMVs in the presence of KYT-1 and KYT-36 (10 μM of each). *p < 0.05. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Genetic Strategies Validation. </b>Caspase-1 but not gasdermin-D processing depends on ASC oligomerization.(a) Western blot analysis for cleaved caspase-1 p20, IL-1 beta p17, and HMGB-1 in cell supernatants (SN) and pro-caspase-1, pro-IL-1 beta and HMGB-1 in cell lysates (lys) of LPS-primed immortalized Asc−/− BMDMs or Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R 3 h after poly(dA:dT) transfection (1 μg ml−1). (b) Release of LDH from LPS-primed immortalized Asc−/− BMDMs or Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R, or derived Casp1 knockouts 3 h after poly(dA:dT) transfection (1 μg ml−1). (c) Western blot analysis for processing of full-length gasdermin-D (GSDMDFL) into the active N-terminal fragment (GSDMDN-term) in combined lysates and supernatants (lys + SN) of LPS-primed immortalized Asc−/− BMDMs expressing ASCFL, ASCY59A or ASCE80R, or derived Casp1 knockouts 3 h after poly(dA:dT) transfection (1 μg ml−1). Arrowhead, gasdermin-DNterm p30; *a cross-reacting band. (d) Release of LDH from LPS-primed primary C57BL/6 WT (WT), Casp1−/−/Casp11−/− or Asc−/− BMDMs infected with S. Typhimurium (multiplicity of infection (MOI)=10, 1 h). (e) Western blot analysis for processing of full-length gasdermin-D (GSDMDFL) into the active N-terminal fragment (GSDMDN-term) in combined lysates and supernatants (lys+SN) of LPS-primed primary C57BL/6 WT (WT), Casp1−/−/Casp11−/− or Asc−/− BMDMs infected with S. Typhimurium (MOI=10, 1 h) or left uninfected. Arrowhead, gasdermin-DNterm p30; *a cross-reacting band; ** a S. Typhimurium-specific cross-reactive band. See also Supplementary Figs 8 and 9. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms11929), licensed under a CC-BY license. Not internally tested by R&D Systems.

Nimbidiol treatment mitigated elevated pro-inflammatory cytokine and chemokine in the diabetic kidney. Representative images from (A) Semi-quantitative RT-PCR analyses showing gene expression and (B) western blot analyses showing protein expression of TNF-alpha , IL-1 beta and MCP-1 in the kidney. The bar diagrams represent the fold change from WT + Saline. Data are mean ± SD (n = 6/group). *p < 0.05 versus WT + Saline, WT + Nimbidiol and Akita + Nimbidiol, †p < 0.05 versus Akita + Saline. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/36522378), licensed under a CC-BY license. Not internally tested by R&D Systems.

Loss IL-1 beta K133 ubiquitylation or the IL-1 beta K133:D129 salt bridge stabilizes IL-1 beta .a WT Il1b, Il1bK133A and Il1bD129A cDNAs were expressed in 293T cells and 48 h after transfection cells exposed to cycloheximide (CHX, 20 μg/ml) for up to 7 h. Cell lysates were immunoblotted for IL-1 beta levels. The arrow indicates pro-IL-1 beta . Actin is included as a loading control. One of three experiments. b WT Il1b cDNA was transfected into 293T cells and after 48 h cells were cultured with MG132 (20 μM) as indicated for 15 min prior to cycloheximide (CHX, 20 μg/ml) treatment for up to 7 h. Immunoblots were performed on total cell lysates for IL-1 beta protein levels. The arrow indicates pro-IL-1 beta . Actin is included as a loading control. One of two experiments. c 293T cells expressing WT IL-1 beta , IL-1 beta K133R, IL-1 beta K133A, IL-1 beta D129A and IL-1 beta D117A were co-transfected with Caspase-1 (250 or 500 ng) in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk (50 μM) for 24 h. Immunoblots were performed on cell supernatants and total cell lysates for the indicated proteins. One of 2–3 experiments. d, e 293T cells expressing WT IL-1 beta , IL-1 beta K133R, IL-1 beta K133A, IL-1 beta S134A (phospho-ablating) and IL-1 beta S134E (phospho-mimetic) were co-transfected with 200 and 400 ng of d Caspase-1 or e Caspase-8, as indicated, for 24 h. IL-1 beta secretion was analyzed by ELISA. Data are the mean + S.D of three independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

Identification of new NLRP3 inflammasome activators. a Schematic of activator-suppressor screens for IL-1 beta release. b Effect of the top 3 activators (6 h treatment, this notation is used throughout the figure legends) on IL-1 beta release (nigericin; positive control). c Immunoblot of biologically active IL-1 beta (p17) and caspase-1 (Casp1; p20) following activator treatment (3 h). Data represent two independent experiments; full gel images are in Supplementary Figure 14. d Effect of gene knockout of NLRP3 inflammasome components on IL-1 beta release (6 h). Guide RNA corresponding to non-targeting negative controls (N.Ctl) or gene-specific targets are indicated. e Effect of activators on LDH release (1 h, 3 h, 6 h). f Effect of P2RX7 antagonist on IL-1 beta release (1 h A438079 pretreatment followed by 6 h activator treatment in the presence of A438079). Data are mean ± s.d.; n = 3 (b, f), 6 (e), or 8 (d) biological replicates from one independent experiment; data are representative of two independent experiments. P values were determined by two-way ANOVA followed by Tukey’s multiple testing (d, e). *P < 0.0001 compared to N.Ctl1 (d) or no activator control of each time point (e). THP-1 macrophages (b, c, e, f) or THP-1 macrophages expressing guide RNA (d) were used for measurements. Activators were used at 1 μM (niclosamide), 2.5 μM (AG-879), 1 μM (ouabain), 20 μM (nigericin), or 5 mM (ATP) (c–f) Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/30740538), licensed under a CC-BY license. Not internally tested by R&D Systems.

Niclosamide-biased suppressors prevent intracellular potassium loss. a Results of suppressor screens using nigericin (20 μM) or niclosamide (5 μM) as activators. Yellow circles indicate the top 60 validated activators. b, c Effect of niclosamide-biased suppressors on IL-1 beta release. d Effect of suppressors on LDH release. e Immunoblot of biologically active IL-1 beta (p17) and caspase-1 (Casp1; p20) after suppressor treatment. Data represent two independent experiments. Full gel images are shown in Supplementary Figure 14. f FACS analysis of mitochondrial ROS. Data represent three independent experiments. g Effect of suppressors on intracellular potassium level. h Rescue of IL-1 beta release by nigericin following suppressor treatment. Numbers (1–3) represent experimental scheme illustrated above the figure panel. Data are mean ± s.d.; n = 3 (b, c), 4 (g), 5 (h), or 6 (d) biological replicates from one independent experiment; data are representative of two independent experiments. P values were determined by two-way ANOVA followed by Tukey’s multiple testing. *P < 0.05, ***P < 0.001, ****P < 0.0001 as indicated. THP-1 macrophages (a–e, h) or THP-1 macrophages expressing NLRP3 guide RNA (f, g) were used for measurements. 1 h suppressor treatment (10 μM DPI, 10 μM LP44, or as indicated) was followed by 1 h (f) or 6 h (b–e, g–h) activator treatment (1 μM niclosamide, 20 μM nigericin, 1 μM ouabain, or 250 μg mL−1 silica) in the presence of suppressors (b–h). Nigericin rescue (h) (20 μM) for 1 h was performed following 6 h niclosamide treatment Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/30740538), licensed under a CC-BY license. Not internally tested by R&D Systems.

Cleavage of NLRP1B is sufficient to promote inflammasome activation.A) 293T cells were transfected with WT, TEV-site2 or TEV-site1 GFP-HA-NLRP1B along with empty vector, TEV expression vector, or a LF expression plasmids. In all conditions cells were also co-transfected with Casp1 and Il1b expression vectors. Cleavage of GFP-HA-NLRP1B and IL-1 beta was determined 24 h post transfection. B) Immortalized B6 macrophages were transduced with a retrovirus encoding the indicated GFP-HA-NLRP1B form followed by a sequential transduction with a TEV-expression retrovirus co-expressing THY1.1. Percent transduction was determined by measuring expression of the respective retroviral integration markers (GFP and anti-THY1.1-PE-Cy7) by flow cytometry, and are expressed in relative fluorescent units (RFU). The numbers within each quadrant represent the percentage of live cells within the respective quadrant. C) RAW264.7 macrophages were transduced with GFP-HA-NLRP1B and a Tet-On construct expressing the indicated gene. Cells were treated with 5 µg/ml doxycycline for 20 h and supernatants were assayed for LDH release. D) 293T cells were transfected with empty vector, FL-NLRP1B-HA, the truncated NLRP1B-HA, or delta LRR HA-NLRP1B, along with Casp1 and Il1b and assayed by immunoblotting. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/23818853), licensed under a CC-BY license. Not internally tested by R&D Systems.

LPS-induced IL-1 beta is increased in Il1bK133R/K133R mice in vivo.a BMDMs derived from mice of the indicated genotypes were treated with LPS (100 ng/ml) for 3 h, followed by proteasome inhibition (PS341, 10 μM) for an additional 3 h. Biotin conjugated IL-1 beta antibody was used to purify IL-1 beta from cell lysates and its modification analyzed using the indicated ubiquitin (Ub) chain-specific antibodies. Cell lysates from the same experiment were run in triplicate with roman numerals on the bottom right of the blots indicating the membrane probed. One of two independent experiments. b BMDMs were primed with LPS (100 ng/ml) for 3 h followed by treatment for a further 3 h with PS341 (10 μM). Cells were subsequently lysed in buffer containing either PS341 (10 μM), or PS341 and PR619 (10 μM), as indicated. Recombinant caspase-1 (5 Units) was added and cell lysates incubated at 37 °C for 80 min. Subsequently, ubiquitylated proteins were purified by Tandem Ubiquitin Binding Entities (TUBEs) and analyzed by immunoblot. One of two independent experiments. c–f WT and Il1bK133R/K133R mice were injected intra-peritoneally with 100 μg of LPS. After 2 h serum and peritoneal fluid was harvested and analyzed by ELISA for levels of c IL-1 beta , d TNF, and e IL-6. Data are mean ± SEM, symbols represent individual mice, n ≥ 19 mice/genotype from three pooled independent experiments for c, d and n ≥ 14 for e from 2 to 3 pooled experiments. P-values were calculated using an unpaired two-tailed t-test for comparisons between genotypes. f Peritoneal fluid was analyzed by immunoblot for the indicated proteins. Each lane represents an individual mouse. n = 5–6 mice per genotype. One of two independent experiments (analysis of nine additional WT and 19 Il1bK133R/K133R mice are shown in Supplementary Fig. 7b). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

PEG-Arg1 treatment reduced retinal inflammation and reversed MΦ/microglia soma enlargement. (A,C) Representative Western blots of the pro-inflammatory cytokines IL-1 beta and TNF- alpha in retinal protein lysates from PEG-A1-treated db/db mice with quantification (B,D, respectively) compared with the PEG-treated db/db controls (n = 6/group, * p < 0.05, ** p < 0.01). (E) Retinal flat-mounts imaged at the level of the superficial vascular plexus labeled with isolectin B4 (vasculature) and Iba-1 (MΦ/microglia) with Iba-1 in the enlarged inset (boxed in white) showing exceptionally enlarged perivascular MΦ/microglia. (F) Quantification of MΦ/microglia soma size, demonstrating significant size reductions in the db/db mice treated with PEG-Arg1 compared with the vehicle-treated controls (n = 3 mice/group, * p < 0.05). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36139465), licensed under a CC-BY license. Not internally tested by R&D Systems.

ATP and nigericin can augment proteasomal-mediated IL-1 beta degradation.a BMDMs were primed with Lipopolysaccharide (LPS, 50 ng/ml) for 3 h and stimulated with ATP (5 mM) and Nigericin (10 μM) for up to 2 h. Total cell lysates were analyzed by immunoblot. One of three experiments. b, cCaspase-1−/− BMDMs were primed with LPS (50 ng/ml) for 3 h, and in the last 30 min of priming treated with MG132 (20 μM), as indicated. Cells were treated with b ATP (5 mM) and c Nigericin (10 μM) for up to 90 min. Immunoblots were performed on total cell lysates to detect the specified proteins. One of three experiments. d WT, Nlrp3−/−, Asc−/− and Caspase-1−/− BMDMs were primed with LPS (50 ng/ml) for 3 h and stimulated with ATP for 20 min. Ubiquitylated (Ub.) proteins were purified from cell lysates by Tandem Ubiquitin Binding Entities (TUBEs). Immunoblots were performed on cell lysates and TUBE isolated ubiquitylated proteins for the indicated proteins. One of two experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

Loss of K133 ubiquitylation in mice stabilizes IL-1 beta and increases IL-1 beta activation.a. WT and Il1bK133R/K133R BMDMs were primed with Lipopolysaccharide (LPS, 50 ng/ml) or Pam3Cys-Ser-(Lys)4 (Pam3Cys, 500 ng/ml) for 3 h with Q-VD-OPh (20 μM) added in the last 30 min of priming. Cells were then treated with the protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml) for up to 6 h. Total cell lysates were analyzed for the indicated proteins by immunoblot. One of four experiments. b, c WT and Il1bK133R/K133R BMDMs were primed with LPS (50 ng/ml) for 3 h and then treated with ATP (5 mM) and Nigericin (10 μM) for the times indicated in Opti-MEM media. b IL-1 beta and c TNF levels were measured in cell supernatants by ELISA. Data are the mean + SD of two independent experiments combined using three mice of each genotype. d WT and Il1bK133R/K133R BMDMs were primed with LPS (50 ng/ml) for 3 h and treated with ATP (5 mM) and Nigericin (10 μM) for up to 60 min in Opti-MEM media. Cell supernatants and lysates were analyzed by immunoblot for the indicated proteins. One of three experiments. e WT and Il1bK133R/K133R neutrophils sorted from bone marrow were primed with IFN gamma (50 ng/ml, 1 h) and then treated with LPS (100 ng/ml) for a further 3 h. Cells were then stimulated with Nigericin (10 μM) for up to 6 h, as indicated. Cell supernatants and lysates were analyzed by immunoblot for the specified proteins. One of three experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33976225), licensed under a CC-BY license. Not internally tested by R&D Systems.

Co-localization of inflammasome components&production of active interleukin-1 beta (IL-1 beta )&IL-18 (IL-18) in the XII. nucleus. (D) Representative WB images of IL-1 beta &IL-18 proteins in of the hypoglossal nuclei after unilateral axotomy of corresponding brain nerve. Arrows indicate pro-forms, arrowheads show active cytokines. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/33328988), licensed under a CC-BY license. Not internally tested by R&D Systems.

EhaF inhibits innate immune responses to EHEC infection.a–c, e–l Secretion of indicated cytokines by C57BL/6 or Tlr4−/− BMDMs infected with EHEC, the indicated isogenic mutant, or complement strains at an MOI of 50 (MOI of 50 for all infections hereafter unless otherwise indicated) or treated with 0.5 μg/ml LPS or 0.5 μg/ml Pam3CSK4 for 6 h (b, c, f, g, h, j, k) or 18 h (a, e, i, l). d Immunoblot for pro IL-1 beta and beta -actin in the lysates of BMDMs infected with EHEC or delta OI-14-15 for the indicated times. m, n Fold increase in the expression of indicated genes in C57BL/6 or Tlr4−/− BMDMs infected with the indicated E. coli strains or treated with 0.5 μg/ml Pam3CSK4 relative to uninfected BMDMs (medium) as determined by real time quantitative PCR at 2 h post-stimulation. o, p Secretion of indicated cytokines by Caco2 cells infected with the indicated (on x-axis) MOI of EHEC or delta EhaF for 24 h. a–c, e–p, Data (mean ± SEM) were from three independent experiments and each dot is a mean of each experiment’s technical replicates. d Immunoblot from one experiment representative of three independent experiments is shown. Statistical significance was assessed using one-way ANOVA (a–c, e–i) or two-way ANOVA (j–p) followed by Tukey’s post-test. p < 0.05 indicated statistical significance. Multiplicity adjusted p values are presented. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37041208), licensed under a CC-BY license. Not internally tested by R&D Systems.

Elevated retinal inflammation and MΦ/microglia soma enlargement in db/db mice. (A) Representative Western blots of IL-1 beta in retinal protein lysates with quantification in (B), demonstrating significant increases in IL-1 beta (n = 10) expression in db/db mice compared with the lean DB/+ controls. (C) Retinal flat-mounts imaged at the level of the superficial vascular plexus and labeled with isolectin B4 (vasculature) and Iba-1 (MΦ/microglia). White arrows point to the perivascular macrophages with Iba-1 in the enlarged inset (boxed in white) showing exceptionally enlarged perivascular MΦ/microglia. (D) Quantification of MΦ/microglia soma size, demonstrating a significant size increase in db/db mouse retinas compared with the lean controls (n = 3 mice/genotype, * p < 0.05). Data are presented as the mean ± SEM. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36139465), licensed under a CC-BY license. Not internally tested by R&D Systems.

Rac1 haploinsufficiency rescues arthritis & inflammatory signaling in Pggt1b delta / delta mice. d WBs showing levels of mature Il-1 beta & caspase-1 in supernatants (Sup), & pro-Il-1 beta & pro-caspase-1 in lysates (Lys) of LPS (200 ng/ml) stimulated BM macrophages; tubulin in lysates was used as a loading control. The antibiotic nigericin (28 mM) was used as a positive control for inflammasome-mediated caspase-1 activation & Il-1 beta production. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31484924), licensed under a CC-BY license. Not internally tested by R&D Systems.

Examining the role of microglia-derived IL-1 beta in promoting astrocyte TIMP-1 expression. A. Determination of IL-1 beta in LPS-activated MG-CM. Microglial cultures were treated with LPS for 2 days, then subject to either six rounds of IL-1 beta immunodepletion, or mock depletion, as described in Materials and Methods, and samples analyzed for the presence of IL-1 beta . Note that IL-1 beta was detected in the LPS-activated MG-CM before, but not after the IL-1 beta immunodepletion, demonstrating the effectiveness of the immunodepletion strategy. B and C. The effect of removing IL-1 beta from LPS-activated MG-CM on astrocyte TIMP-1 production. Pure astrocyte cultures received either: LPS, microglia + LPS, LPS-activated MG-CM (mock depleted) or LPS-activated MG-CM depleted of IL-1 beta . Note that the presence of LPS-activated microglia or LPS-activated MG-CM strongly induced TIMP-1 expression in astrocytes, but this effect was markedly attenuated after IL-1 beta removal from the MG-CM. * p < 0.02. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/21631912), licensed under a CC-BY license. Not internally tested by R&D Systems.

AGER is required for caspase-11 inflammasome activation. (A–C) Analysis of cytotoxicity (A), IL-1 beta release (B), and IL-18 release (C) in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) in the absence or presence of FPS-ZM1 (100 nM, 500 nM, and 1 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05 vs. HHD group, t test. (D–F) Analysis of cytotoxicity (D), IL-1 beta release (E), and IL-18 release (F) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h), LPS electroporation (LPSe; 1 μg, 16 h), or E. coli infection (MOI = 25, 16 h). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05 vs. WT group, t test. (G) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h). Western blot data represent two independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31440260), licensed under a CC-BY license. Not internally tested by R&D Systems.

GSDMD is required for nuclear DAMP complex-induced pyroptosis. (A) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in indicated LPS-primed BMDMs after HHD treatment (500 ng/mL, 16 h). (B–G) Analysis of cytotoxicity (B), IL-1 beta (C), IL-18 (D), TNF (E), IL-6 (F), and IL-12 (G) release in indicated LPS-primed BMDMs after HHD treatment (500 ng/mL, 16 h) in the absence or presence of the overexpression of GSDMD WT or D275A cDNA. n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. Western blot data represent two independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31440260), licensed under a CC-BY license. Not internally tested by R&D Systems.

Charged liposomes activate the NLRP3 inflammasome to induce IL-1 beta secretion ELISA for IL-1 beta from the supernatants of (a) LPS-primed wild-type BMDMs or (b) PMA-primed THP1 cells that were stimulated with either the indicated liposomes (30 μg/ml for BMDMs and 50 μg/ml for THP1 cells) or alum (250 μg/ml for BMDMs and 500 μg/ml for THP1 cells). (c) Immunoblots of procaspase-1, activated caspase-1 (p10), pro-IL-1 beta and cleaved IL-1 beta (p17) in the culture supernatants (Sup) and cell lysates (Lys) from LPS-primed BMDMs after stimulation with indicated liposomes or alum. (d) IL-1 beta from supernatants of LPS-primed BMDMs that were pretreated with caspase-1 inhibitor (z-YVAD-fmk, 10 μM) for 45 min followed by stimulations with indicated liposomes or alum. (e) ELISA for IL-1 beta from the supernatants of LPS-primed or unprimed wild-type BMDMs that were stimulated with indicated liposomes (30 μg/ml) or alum (250 μg/ml). (f) The IL-1 beta levels from the supernatants of LPS-primed immortalized mouse macrophages from wild-type, Nlrp3−/−, Asc−/−, Caspase-1−/− or Nlrc4−/− mice that were stimulated with liposomes (70 μg/ml). (g) ELISA for IL-1 beta from the supernatants of LPS-primed wild-type BMDMs that were cultured in 150 mM of KCl or NaCl followed by stimulation with liposomes or alum. Data in a, b and d–g are shown as mean ± s.d., and all data are representative of at least three independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/23511475), licensed under a CC-BY license. Not internally tested by R&D Systems.

AGER-mediated lipid peroxidation promotes caspase-11 inflammasome activation. (A–C) Analysis of lipoxygenase activity (A), MDA level (B), and 4-HNE level (C) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h) in the absence or presence of FPS-ZM1 (1 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. (D–F) Analysis of cytotoxicity (D), IL-1 beta release (E), and IL-18 release (F) in indicated LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h) in the absence or presence of zileuton (5 μM). n = 3, data expressed as means ± SD of three independent experiments, *P < 0.05, t test. (G) Western blot analysis of indicated proteins in the supernatant (SN) or cell lysate in LPS-primed BMDMs after HHD treatment (500 ng/ml, 16 h) or LPS electroporation (LPSe; 1 μg, 16 h). Western blot data represent two independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31440260), licensed under a CC-BY license. Not internally tested by R&D Systems.

IL-1 beta promotes priming and activation of the NLRP3 inflammasome in HUVEC. The expression of the NLRP3 inflammasome components (A) NLRP3 and (B) pro-IL-1 beta was determined by Western blot in total lysates from HUVEC stimulated for 18-24 h with IL-1 beta . Representative blots are shown on top of the corresponding graphs, where beta -Actin was employed as loading control (n= 4-5; *p<0.05 vs. untreated cells by unpaired t-test). (C) The NLRP3 inflammasome activation was determined by the observation of ASC specks by indirect immunofluorescence after treatment with IL-1 beta alone or in presence of MCC 950 (1 µM). The percentage of ASC specks per cell culture field was quantified under a fluorescence microscope (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). (D) Confocal representative photographs (630X magnification) where white arrowheads show the location of toroidal-shaped ASC specks as detected by a specific antibody against ASC (red), while cell nuclei were counterstained with DAPI (blue). (E) IL-1 beta , the final product of the NLRP3 inflammasome activation was measured in the supernatant of treated cells by ELISA (n= 4-5; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cultures levels by two-way ANOVA and Tukey post-hoc test). HUVEC were treated with 2.5 ng/ml IL-1 beta in all experiments. All data are shown as mean ± SEM and expressed as percentage of IL-1 beta -induced levels. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

Manoalide inhibits the NLRP3 inflammasome activation. (A-C) BMDMs were primed with LPS for 3 h and then were pretreated with 125, 250, 500 nM manoalide before stimulation with nigericin for 15 min (A) ELISA of mature IL-1 beta in supernatant from BMDMs. (B) Western blots analysis of supernatant (SN) and cell lysates (Input) from BMDMs. (C) LDH release in the supernatant from BMDMs. (D) ELISA of mature IL-1 beta and (E) Western blots of SN and Input from BMDMs primed with LPS and stimulated with nigericin, MSU and ATP with or without 500 nM manoalide. (F-H) BMDMs were primed with Pam3K for 3 h and then were pretreated with 125, 250, 500 nM manoalide before stimulation with cytosolic LPS (cLPS) for 16 h. (F) Production of mature IL-1 beta in supernatant from BMDMs. (G) Western blots analysis of supernatant (SN) and cell lysates (Input) from BMDMs. (H) LDH release in the supernatant from BMDMs. (I) ELISA of mature human IL-1 beta in supernatant from PBMCs primed with LPS for 3 h and then were pretreated with 125, 250, 500 nM manoalide before stimulation with nigericin. Data are derived from three independent experiments (A,C,D,F,H,I) and displayed as mean and SEM (n = 6) or represent three independent experiments (B,E,G). Statistically significant difference was calculated by unpaired Student’s t-test: ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35252186), licensed under a CC-BY license. Not internally tested by R&D Systems.

The activation of NLRP3 by IL-1 beta is mediated by NF-kappa B. The expression of (A) phosphorylated p65 (p-p65) and total p65 (p65), used as a marker of NF-kappa B activation, and the NLRP3 inflammasome components (B) NLRP3, (C) pro-IL-1 beta and (D) mature IL-1 beta levels was determined by Western blot in total lysates from HUVEC stimulated for 18 h with IL-1 beta in presence or absence of PDTC (100 µM). Representative blots are shown on top of the corresponding graphs, where beta -Actin was employed as loading control (n= 5-7; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). All data are shown as mean ± SEM and expressed as percentage of IL-1 beta -induced levels. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

PL dose-dependently inhibits nigericin-induced NLRP3 inflammasome activation. (A) Cell viability of PL (1–40 μM) in peritoneal macrophages. (B–D) ELISA of IL-1 beta (B), TNF-alpha (C), and release of LDH (D) in supernatants from LPS-primed mouse peritoneal macrophages treated with 1–10 μM PL and stimulated with nigericin. (E) Immunoblot of supernatants or cell lysates from LPS-primed mouse peritoneal macrophages treated with 1–10 μM PL and stimulated with nigericin. All data were representative of three independent experiments. Values shown are mean ± SD. For statistical analysis, A–D were analyzed using one-way ANOVA and the Bonferroni test. *p < 0.05; **p < 0.01; ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35095532), licensed under a CC-BY license. Not internally tested by R&D Systems.

Manoalide has no effects on the activation of AIM2 inflammasome, IPAF inflammasome or Pyrin inflammasome. (A) ELISA of mature IL-1 beta and (B) Western blots of SN and Input from BMDMs primed with LPS and stimulated with nigericin, Poly (A:T) or salmonella (MOI = 10) with or without 500 nM manoalide. (C) ELISA of mature IL-1 beta in SN from BMDMs primed with LPS and then were pretreated with 500 nM manoalide before stimulation with C3 toxin. Data are derived from three independent experiments (A,C) and displayed as mean and SEM (n = 6) or represent three independent experiments (B). Statistically significant difference was calculated by unpaired Student’s t-test: ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35252186), licensed under a CC-BY license. Not internally tested by R&D Systems.

149-01 does not affect the activation of AIM2, NLRC4, or Pyrin inflammasomes. (A–C) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, poly (A/T), Salmonella or C3 toxin. (A,C) IL-1 beta releases in supernatants were detected by ELISA. (B) Active IL-1 beta and p20 in supernatants and pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m (n = 6) (A,C), or are representative of three independent experiments (B). One-way ANOVA was applied to calculate statistical significance: ***p <.001, NS, not significant. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35250572), licensed under a CC-BY license. Not internally tested by R&D Systems.

Identification of 149-01 as a highly potent inhibitor for NLRP3 inflammasome. (A) 149-01 structure. (B–F) BMDMs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 (200-600 nM) for 30 min, lastly stimulated with nigericin. (B) IL-1 beta releases in supernatants were detected by ELISA. (C) Active IL-1 beta and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates (Input) were measured by western blot. (D) The release of LDH in supernatants. (E) IL-6 and (F) TNF-alpha secretion levels in supernatants were determined by ELISA. (G,H) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, ATP or MSU. (G) IL-1 beta releases in supernatants were detected by ELISA. (H) Active IL-1 beta and p20 in supernatants and pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates were measured by western blot. (I,J) PBMCs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 for 30 min, lastly stimulated with nigericin. (I) IL-1 beta and (J) TNF-alpha secretion levels in supernatants were determined by ELISA. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m (n = 6) (B,D–G,I,J), or are representative of three independent experiments (C,H). One-way ANOVA was applied to calculate statistical significance: ***p <.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35250572), licensed under a CC-BY license. Not internally tested by R&D Systems.

FA inhibits I/R-induced myocardial injury by inhibiting NLRP3 inflammasome activation. (A–F) Expression of NLRP3, pro-caspase-1, caspase-1, pro-IL-1 beta , and IL-1 beta in the heart tissue of WT mice was detected by western blotting. (G) HE staining of heart tissue sections in WT mice and Nlrp3−/−mice. (H–K) Expression of IL-1 beta , caspase-1, and NLRP3 in heart tissues of WT mice and Nlrp3−/− mice in each experimental group, detected by western blotting. (L–O) Expression of CK-MB, LDH, troponin-1, and NT-proBNP in the serum of each experimental group in WT mice and Nlrp3−/−mice, determined by ELISA. Data are expressed as the means ± sd from three independent experiments or are representative of three independent experiments. **p < 0.01, ***p < 0.001; ns indicates no significance. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35496295), licensed under a CC-BY license. Not internally tested by R&D Systems.

PL inhibits ATP or MSU-induced NLRP3 inflammasome activation. (A–C) ELISA of IL-1 beta (A), TNF-alpha (B), and release of LDH (C) in supernatants from LPS-primed mouse peritoneal macrophages treated with 10 μM PL or DMSO and stimulated with ATP or MSU. (D) Immunoblot of supernatants or cell lysates from LPS-primed mouse peritoneal macrophages treated with 10 μM PL and stimulated with indicated stimuli. All data were representative of three independent experiments. Values shown are mean ± SD. For statistical analysis, A, B, and C were analyzed using two-way ANOVA and the Bonferroni test; **p < 0.01; ***p < 0.001; ****p < 0.0001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35095532), licensed under a CC-BY license. Not internally tested by R&D Systems.

The inflammasome NLRP3 is implicated in the pro-senescence effect of IL-1 beta in endothelial human cells. HUVEC were treated with IL-1 beta (2.5 ng/ml) alone or in presence of the NLRP3 inflammasome inhibitor MCC 950 (1 μM) for 18-24 h. (A) SA-beta -Gal positive stained cells were quantified by manual scoring by a blind observer after the indicated treatments. (B) Representative phase-contrast images are shown SA-beta -Gal positive cells stained in blue (black arrowheads). Data are expressed as percentage of the number of senescent cells stained in non-treated cells (n= 5-7; *p<0.05 vs. untreated cells; #p<0.05 vs IL-1 beta -treated cultures levels by two-way ANOVA and Tukey post-hoc test). (C) The expression of p21 was determined by Western blot in total lysates from HUVEC. A representative blot is shown on top of each graph, where beta -Actin was employed as loading control (n= 6-8; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). All data are shown as mean ± SEM. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

Protein expression of inflammasome NLRP3 in PBMCs in the presence of Convulxin. Western blot of NLRP3, Pro-Caspase-1, Active Caspase-1, IL-1 beta , and beta -actin (control) using 1 × 107 PBMCs stimulated RPMI (negative control), LPS (1 μg/mL; positive control) or CVX (5 and 10 μg/mL) for 3 h. (A) Figure representative of one experiment of three independent experiments. Relative immunoreactivity analysis (fold of beta -actin) of the western blots from NLRP3, Pro-Caspase-1, Active Caspase-1, IL-1 beta (B). IL-1 beta release was also quantified in PBMCs pretreated with N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (30 µM), Ac-YVAD-cmk (50 µM) (C), MCC950 (10 µM) (D) for 30 min and after incubated with RPMI (negative control) or CVX (10 µg/mL). IL-1 beta was also quantified in PBMCs pretreated with Apocynin (300 µM) and Rotenone (10 µM) for 30 min and after incubated with RPMI (negative control) or CVX (10 µg/mL) (E). Results were expressed in pg/mL of released cytokines and represent the mean ± S.E.M of 3 donors. Values are mean S.E.M. from 3 donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to negative control (data were presented with ANOVA followed by Dunnett post-test). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35304541), licensed under a CC-BY license. Not internally tested by R&D Systems.

149-01 targets Cys409 of NLRP3. HEK-293T cells transfected with Flag-tagged NACHT (A), LRR (B), PYD (C), or NACHT (C409A) (D) were lysed and the cell lysates incubated overnight with or without 149-01 (80 μM) before pronase digestion were detected by western blot. (E) The interactions between NEK7 and WT or mutant NLRP3 in HEK-293T cells treated with or without 149-01 (3 μM) were evaluated by IP and western blot. (F,G)Nlrp3−/− BMDMs reconstituted with mouse WT or mutant NLRP3 were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin. (F) IL-1 beta releases in supernatants were detected by ELISA. (G) Active IL-1 beta and p20 in supernatants and NLRP3, pro-IL-1 beta , pro-caspase-1 and beta -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m (n = 6) (F), or are representative of three independent experiments (A–E,G). One-way ANOVA was applied to calculate statistical significance: ***p <.001, NS, not significant. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35250572), licensed under a CC-BY license. Not internally tested by R&D Systems.

AMPK activation restores A beta 42‐induced mitochondrial hyperfission and inflammasome activation in LPS‐primed microglia. LPS‐primed microglia were pretreated with AICAR (1 mM) for 1 h and then treated with A beta 42 for 24 h. (a–e) Representative immunoblot (a) and densitometric quantifications (b–e) of the protein levels of pAMPK, AMPK, pSyk, Syk, Drp1, pro‐IL‐1 beta , and beta ‐actin in total cell lysate. (f–g) MitoSOX (red) and DAPI (blue) staining and quantification of MitoSOX fluorescence intensity. Scale bar: 10 μm. (h) Immunostaining of ASC (red) and DAPI (blue). Scale bar: 10 μm. (i) Percentages of microglia containing ASC foci were counted. Data are shown as mean ± SEM from at least three independent experiments and are analyzed by one‐way ANOVA and Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35474599), licensed under a CC-BY license. Not internally tested by R&D Systems.

Ang-(1-7) attenuates the induction and activation of the NLRP3 inflammasome mediated by IL-1 beta in HUVEC. The expression of NLRP3 inflammasome components (A) NLRP3 and (B) pro-IL-1 beta was determined by Western blot in total lysates from HUVEC stimulated for 18-24 h with IL-1 beta (2.5 ng/ml) in presence or absence of Ang-(1-7) (100 nM). Representative blots are shown on top of the corresponding graphs, where beta -Actin was employed as loading control (n= 6-8; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). (C) The NLRP3 inflammasome activation was determined by the detection of ASC specks by indirect immunofluorescence. The percentage of ASC specks per culture field was quantified under a fluorescence microscope (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). (D) Confocal representative photographs (630X magnification) where white arrowheads show the location of toroidal-shaped ASC specks as revealed by a specific antibody against ASC (red), while cell nuclei were counterstained with DAPI (blue). (E) levels of mature IL-1 beta were measured in total cell lysates by Western blot, and also (F) detected in the supernatants of treated cells by ELISA (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1 beta -treated cells, by two-way ANOVA and Tukey post-hoc test). All data are shown as mean ± SEM and expressed as percentage of IL-1 beta -induced levels. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35111374), licensed under a CC-BY license. Not internally tested by R&D Systems.

miR-1208 is the key miRNA bind to METTL3 in EVs to exert anti-inflammatory effects. A. Western blot analysis of Ago-2 expression in hucMSCs lysates. B. Western blot analysis of IL-1 beta and cleaved caspase-1 levels in culture SN and pro-IL-1 beta , pro-caspase-1, NLRP3, and METTL3 levels in macrophage cell lysates in each group. C. Heat map of miRNA levels in hucMSCs-EVs (n = 3, Student’s t test). D. Four most significantly upregulated miRNAs in hucMSCs-EVs. E. The target sequence of miR-1208 estimated within 3′-UTR of METTL3. F. Western blot analysis of IL-1 beta and cleaved caspase-1 levels in culture SN and pro-IL-1 beta , pro-caspase-1, NLRP3, and METTL3 levels in macrophage cell lysates in each group. G. Luciferase reporter assay showing that METTL3 is the target gene for miR-1208 (n = 3, Student’s t test) Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35842714), licensed under a CC-BY license. Not internally tested by R&D Systems.

Multiple TCAs specifically induce the NLRP3 inflammasome activation by triggering upstream signaling events. a The structures of these antidepressants. b LPS-primed BMDMs were stimulated with protriptyline, amitriptyline, nortriptyline, imipramine, doxepin, clomipramine, agomelatine, mirtazapine, and phenothiazine. Western blot assessed the expression of caspase-1 and IL-1 beta in SN and GSDMD cleavage in WCL. c BMDMs were incubated with LPS and MCC950 and then treated with imipramine, amitriptyline, protriptyline and nortriptyline. The expressions of IL-1 beta and caspase-1 in SN as well as GSDMD cleavage in WCL were evaluated using western blotting. dNlrp3−/− BMDMs were incubated with LPS followed by imipramine, amitriptyline, protriptyline and nortriptyline stimulation. The expression of IL-1 beta and caspase-1 in SN and GSDMD cleavage in WCL were measured by western blotting. e BMDMs or hepatocytes were pretreated with LPS and MCC950 and then treated with nortriptyline, western blot was used to measure the expression of caspase-1 in SN. f BMDMs or Kupffer cells were incubated with LPS and MCC950 followed by nortriptyline treatment. The expression of caspase-1 in SN were assessed by western blot Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37231437), licensed under a CC-BY license. Not internally tested by R&D Systems.

IL‐1 beta secretion occurs independently of pore formationWild‐type and GsdmD−/− HoxB8 macrophages were treated as indicated for 16 h. Supernatants were analyzed for IL‐1 beta secretion by ELISA.HoxB8 macrophages were seeded in an 8‐well Ibidi chamber and treated for 14 h with LPS and GM‐CSF. Cells were fixed and stained for gasdermin D (green), IL‐1 beta (red), and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope.HoxB8 macrophages were co‐stimulated with three different PEG 3 mM for 16 h as indicated. Supernatants were analyzed for IL‐1 beta secretion by ELISA.HoxB8 macrophages were seeded in a 96‐well microplate and treated as indicated. Before treatment, SYTOX green was added and cells were imaged in an IncuCyte incubator microscope every hour for 16 h. Shown is the SYTOX green signal normalized to the total cell area.Wild‐type and GsdmD−/− HoxB8 macrophages were treated as indicated for 16 h. Proteins were extracted and analyzed for IL‐1 beta ubiquitination by western blot.Data information: n ≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one‐way ANOVA with multiple comparisons, or two‐way ANOVA to compare across genotypes. P‐values are shown. Microscopy pictures and westerns are representative images from three biological replicates. Scale bars in microscopy images represent 10 µm.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35695080), licensed under a CC-BY license. Not internally tested by R&D Systems.

NLRP3 inflammasome activation in MSC-mediated mandibular healing. Two-month-old WT or Prx1-Cre/ROSAnTnG mice used, as specified in the figure legends. Mice received mandibular osteotomy surgery & sacrificed at various time points during healing, N=4. (F-H) M-MSCs from WT mice cultured & stimulated with or without 10 μg/ml LPS ± 5 mM ATP during culture, as indicated in the figures. (F) WB of cell lysates & supernatants from M-MSCs cultures. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36778113), licensed under a CC-BY license. Not internally tested by R&D Systems.

GM‐CSF induces NLRP3‐dependent IL‐1 beta secretionBMDMM were treated as indicated with either 60 ng/ml ultra‐pure (UP) LPS, 50 ng/ml human‐derived recombinant GM‐CSF, 40 µM WEHD‐FMK for 16 h, or 5 µM nigericin for the last hour. Supernatants were then analyzed for IL‐1 beta secretion by ELISA.HoxB8 macrophages were treated with the indicated dilutions of GM‐CSF using supernatants from GM‐CSF‐expressing cells. Shown are ELISA analysis of IL‐1 beta secretion in supernatants and western blots indicating IL‐1 beta expression in lysates.HoxB8 macrophages of the indicated genotypes were treated as indicated with 60 ng/ml UP‐LPS, 1% GM‐CSF containing supernatant (approximately 2 ng/ml GM‐CSF), 40 µM WEHD.FMK, and 0.5 µM CRT‐0066101 for 16 h; 5 µM nigericin was added for the last hour. Supernatants were analyzed by ELISA for IL‐1 beta . Inlay panels show western blots for levels of IL‐1 beta expression in the indicated knockouts.HoxB8 cells were treated with 60 ng/ml of UP‐LPS, 1% GM‐CSF supernatant, 50 mM KCl, and 5 µM nigericin as indicated for 16 h before supernatants were analyzed for IL‐1 beta by ELISA. Data information: n ≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one‐way ANOVA with multiple comparisons or two‐way ANOVA to compare across genotypes. P‐values are shown. Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35695080), licensed under a CC-BY license. Not internally tested by R&D Systems.

A 1-hydroxy phenothiazinium-based small molecule inhibits OMV-mediated cytotoxicity and cytokine release in vitro.A Scheme showing the screening strategy to identify potent inhibitors of caspase-11 signaling; B Drug-like properties of PHZ-OH; C Cytotoxicity and IL-1 alpha secretion of wild type (WT), Casp11−/−, Gsdmd−/−, or Nlrp3−/− mouse peritoneal macrophages under a treatment of PHZ-OH (5 μM), and/or a challenge of OMV (10 μg/mL) or E. coli (a MOI of 10) for 16 h; D Caspase-11 mRNA level and cytotoxicity of macrophages primed using LPS (1 μg/mL), Pam3cSK4 (1 μg/mL) or Poly(I:C) (1 μg/mL) for 3 h and challenged with OMV (10 μg/mL) or E. coli (a MOI of 10) for 16 h in the presence or absence of PHZ-OH (5 μM); E TLR4-dependent cytokines (TNF alpha and IL-6) released from WT or Casp11−/− macrophages under a treatment of saline or PHZ-OH (5 μM), and/or a challenge of OMV (10 μg/mL) for 16 h; F Western blots for IL-1 alpha , IL-1 beta and caspase-11 expression and GSDMD cleavage in saline-treated or OMV-challenged macrophages under a treatment of saline or different doses of PHZ-OH (P, 5 or 10 μM); G–J LDH (G or I) and IL-1 alpha as well as IL-1 beta (H or J) released from mouse peritoneal macrophages (WT and Casp11−/−) or THP-1 cells (Transfected with control or caspase-4 siRNA), respectively, in the presence of different concentrations of PHZ-OH (1, 2, 4 μM) and challenged with OMV (10 μg/mL) for 16 h. Data are shown as mean ± SEM of three independent experiments. *P < 0.05. ns, not significant. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35982051), licensed under a CC-BY license. Not internally tested by R&D Systems.

Apilimod mediates inflammasome activation through mitochondrial damage and reactive oxygen species production. (A) Analysis by confocal microscopy of LPS-primed BMDMs stimulated with nigericin or apilimod and subsequently stained with MitoTracker Red (50 nM) follow by the DNA-binding dye DAPI (blue). Scale bar, 20 μm. (B) Analysis by FACS of THP1 cells stimulated with nigericin or apilimod and subsequently stained with MitoTracker deep red (50 nM) and MitoTracker green (50 nM). (C, D) Analysis by confocal microscopy and FACS of BMDMs stimulated with nigericin or apilimod and subsequently stained with MitoSOX Red (1 μM). Scale bar, 20 μm. (E, F) LPS-primed BMDMs were pretreated with a ROS inhibitor (MnTBAP, 150 μM) for 30 min and then stimulated with apilimod for 2 hours. (E) ELISA of IL-1 beta in culture supernatants. (F) Western blot of IL-1 beta , cleaved caspase-1 in supernatants (SN) and pro-IL-1 beta , pro-caspase-1, beta –actin in cell lysate (Input). The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 3 or 4) or are representative of three independent experiments. One-way ANOVA was applied to calculate statistical significance: ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37359517), licensed under a CC-BY license. Not internally tested by R&D Systems.

Apilimod damage the mitochondria through inducing lysosomal calcium flux. (A, B) BMDMs were first primed with LPS for 3 hours, then pretreated with BAPTA-AM (25 μM, 50 μM) for 30 minutes, and stimulated with apilimod for 2 hours. (A) Fluo-4 AM (5 μM) staining of BMDMs after apilimod treatment, FACS analysis was conducted to show changes in cell calcium flux. (B) ELISA of IL-1 beta in culture supernatants. (C–E) LPS-primed BMDMs pretreated with ML-SI3 for 30 min and then stimulated with apilimod. (C) Fluo-4 AM staining of BMDMs after apilimod treatment, FACS analysis was conducted to show changes in cell calcium flux. (D) ELISA of IL-1 beta in culture supernatants.(E) Western blot of IL-1 beta , cleaved caspase-1 in supernatants (SN) and pro-IL-1 beta , pro-caspase-1 (pro-casp1), beta –actin in cell lysate. (F, G) BMDMs transfected with siRNA against Trpml1 or control siRNA were primed with LPS for 3 h and were then treated with apilimod or nigericin. The supernatant of the cell culture was collected. (F)Trpml1 mRNA in BMDMs. (G) ELISA of IL-1 beta in the supernatants of BMDMs. (H) LPS-primed THP-1 cells were pretreated with BAPTA-AM (50 μM) or ML-SI3 (40 μM) for 30 minutes, and stimulated with apilimod for 2 hours. subsequently stained with MitoTracker deep red (50 nM) and MitoTracker green (50 nM). Analysis by FACS of THP1 cells. The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 3) or are representative of three independent experiments. One-way ANOVA was applied to calculate statistical significance: **P < 0.01; ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37359517), licensed under a CC-BY license. Not internally tested by R&D Systems.

Apilimod induces caspase-1 activation and IL-1 beta secretion. (A–E) BMDMs were first primed with LPS for 3 h, then stimulated with doses of apilimod (2.5-7.5 μM) for 2 hours. The supernatant of the cell culture was collected. (A) ELISA of IL-1 beta releases in culture supernatants. (B) Immunoblot analysis of Active IL-1 beta and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1 beta , pro-caspase-1 (pro-casp1) and beta -actin in cell lysates (Input). (C) The release of LDH in supernatants (D) TNF-alpha and (E) IL-6 secretion levels in supernatants were detected by ELISA. (F) Immunoblot analysis of cleaved IL-1 beta and caspase-1 (p20) in culture supernatants (SN) and inactive precursor molecule (pro– IL-1 beta , pro–caspase-1(pro-casp1) in cell lysates (Input) of PMA-differentiated THP-1 cells stimulated with different doses of apilimod. (G) IL-1 beta secretion levels of PBMCs in supernatants were determined by ELISA. The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 4 or 3) or are representative of three independent experiments. One-way ANOVA was applied to calculate statistical significance: ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37359517), licensed under a CC-BY license. Not internally tested by R&D Systems.

MafB knockdown promotes the activation of the NLRP3 inflammasome.a–c PMs were transfected with 50 nM control or MafB siRNAs. Forty-eight hours after transfection, were treated with 100 ng/ml LPS for 3 h. were then washed & stimulated with or without ATP (5 mM), Nigericin (5 uM) or MSU (1 mM) in serum-free media for an additional 1 h. Levels of the supernatant IL-1 beta (a) & IL-18 (b) were determined by ELISA. Levels of the supernatant LDH (c) were determined by LDH assay. d PMs were transfected, primed & stimulated with the NLRP3 agonists as in (a). Supernatant proteins were precipitated by TCA & combined with the respective cellular extracts. Levels of the indicated proteins were determined by WBting. Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. e, f PMs were transfected with control siRNAs or MafB siRNAs. Forty-eight hours after transfection, were primed with LPS for 3 h, followed by stimulation with or without Nigericin for 1 h. were then fixed with 4% PFA & subjected to immunofluorescence staining for ASC. Scale bars: 100 μm (e). ASC specks in the were quantified (f). n = 3–8. g, h J774A.1 macrophages were transfected with control siRNAs or MafB siRNAs. 48 h after transfection, were primed with LPS for 4 h, followed by stimulation with or without ATP (5 mM) or Nigericin (10 μM) for 1 h. Levels of the supernatant IL-1 beta & LDH were determined (g). Levels of the indicated proteins in the supernatants & were determined by WBting (h). Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. Mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. Representative of three independent experiments. LDH lactate dehydrogenase. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37845329), licensed under a CC-BY license. Not internally tested by R&D Systems.

NPC86 reduces IL-1 beta protein levels. (a) An automated Western blot analysis using JESS was performed on adipose tissue from DIOD and DIOD + NPC86 (500 ng/kg b.w.) mice using antibodies against IL-1 beta and GAPDH. Representative blots and quantification are displayed. A statistical analysis was performed in GraphPad Prism using an unpaired t-test (n = 3 per group). The data are presented as mean ± SEM, with statistical significance indicated as *** p < 0.001. (b) The serum concentrations of IL-1 beta , GM-CSF, G-CSF, and MCP-1 were measured by ELISA in DIOD and DIOD + NPC86 (500 ng/kg and 1 μg/kg b.w.) mice. A statistical analysis was performed in GraphPad Prism using an unpaired t-test (n = 3 per group). The data are presented as mean ± SEM, with statistical significance indicated as * p < 0.05, ** p < 0.01. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/40332318), licensed under a CC-BY license. Not internally tested by R&D Systems.

AP specifically reduces NLRP3 inflammasome activation. (a,b) Cell viability assays of J774A.1 cells (a) and differentiated THP-1 cells (b) treated with AP (10 μg/mL, 50 μg/mL, 100 μg/mL) for 2 h. Cell viability was analyzed by EZ-CYTOX, measured at a wavelength of 450 nm. Ns not significant. (c–e) Western blots of IL-1 beta in the supernatants (Sup), pro-IL-1 beta and alpha -tubulin in the soluble lysates (Lys) of treated cells. LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM) (c) or ATP (5 mM) (d) with or without MCC950, differentiated, and LPS-primed THP-1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM) with or without MCC950 (e). (f,g) LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 3 h with transfection of dsDNA (2 μg/mL) (f) or flagellin (1.25 μg/mL) (g). Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41598-024-55651-z), licensed under a CC-BY license. Not internally tested by R&D Systems.

The CR3-Als3 complex at the F-actin cuff promotes Syk phosphorylation and inflammasome activation. A Immunoblot analysis of p-Syk from C57BL/6 and CD18−/− M0 BMDM (without GM-CSF priming) lysates after stimulation with C. albicans hyphae for indicated periods. Total Syk served as a loading control. b WT M0 BMDMs were pretreated with anti-CD11b blocking antibody (M1/70, 10 μg/mL) for 30 min before stimulation with C. albicans hyphae. Cell lysates were subjected to immunoblot analysis of p-Syk and total Syk. IgG2b, kappa was used as an isotype control. c Immunoblot analysis of p-Syk and total Syk from C57BL/6 or CD18−/− M0 BMDMs after WT, als1 delta / delta als3 delta / delta , or als1 delta / delta als3S170Y hyphal infection. d Immunoblot analysis of p-Syk from C57BL/6 and CD18−/− M0 BMDM (without GM-CSF priming) lysates after stimulation with 15 µg ml-1 Als3 or Als3S170Y for indicated periods. Total Syk served as a loading control. e Immunofluorescence images of M0 BMDMs after stimulation with C. albicans hyphae expressing TagBFP for 20 min. p-Tyr, CD11b, and F-actin were analyzed. Scale bars, 7.5 µm. f Immunofluorescence images of M1 BMDMs after infection with C. albicans hyphae expressing TagBFP for 20 min. p-Syk, CD11b, and Dectin-2 were analyzed. Scale bar, 7.5 µm. g Immunoblot analysis of caspase-1 and IL-1 beta after infection with C. albicans hyphae in BMDMs with or without Cyto D and Lat B. Hyphae were induced in RMPI medium a–c, e, f and SC medium plus GlcNAc g. Each experiment was repeated three times independently. Data in g are presented as mean ± SEM. P values in g were determined by one-way ANOVA (Tukey’s multiple comparisons test). ns not significant. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41467-024-48093-8), licensed under a CC-BY license. Not internally tested by R&D Systems.

Inhibition of Clec7a effectively mitigates microglial synaptic phagocytosis and neuroinflammation in PS19 mice. (A) Representative Western blots of indicated proteins in the ventral hippocampal lysates from 6-month-old WT mice and Laminarin-administered PS19 mice. (B) Quantitation of Clec7a, P-SYK, NLRP3, ASC, Procaspase-1, and IL-1 beta proteins in (A) (n = 6 mice per group, unpaired Student’s t test). (C) Representative Western blots of indicated proteins in the ventral hippocampal lysates from 6-months old WT mice and Laminarin-administered PS19 mice. (D,E) Quantitation of PSD95 (D) and synaptophysin (E) proteins (n = 6 mice per group, unpaired Student’s t test). (F) Confocal images of synaptic puncta engulfed by microglia showing the colocalization of PSD95 (green) with CD68 (grays) within Iba1+ microglia (red) in the DG regions of hippocampal tissue from WT mice and Laminarin-administered PS19 mice. Scale bars, 5 μm. (G) Quantitation of CD68 volume. (H) Quantitation of PSD95 engulfment within CD68+ microglial structures (n = 6 mice per group, one-way ANOVA test). * p < 0.05; ** p < 0.01; *** p < 0.001. Data are presented as mean ± SD. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/40243488), licensed under a CC-BY license. Not internally tested by R&D Systems.

Pyroptosis is the primary mode of AEC death induced by cadmium. MLE12 cells were treated with different concentrations of CdCl2 for 24 h. Cell morphology was photographed through a microscope (A, bar = 100 μm). Cell viability was detected by CCK8 assay (B, n = 5). The LDH activity of the cell culture supernatant was assayed (C, n = 3). The Pan-caspase inhibitor (ZVAD-FMK) but not the necroptosis or ferroptosis inhibitor restored cell viability within 24 h of CdCl2 incubation (D, n = 5). Cell morphology was photographed through a microscope (E, bar = 100 μm). The expressions of SPC, NLRP3, pro-Caspase1, Caspase1 p20, IL-1 beta p17, and GSDMD-N protein in MLE12 cells were detected using western blot (F-G, n = 3). The fluorescence intensity of GSDMD was detected by immunofluorescence (H, bar = 5 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/39587603), licensed under a CC-BY license. Not internally tested by R&D Systems.

Hectd3 deficiency promotes the activation of NLRP3 inflammasome.A–H BMDMs isolated from Hectd3+/+ and Hectd3−/− mice were primed with LPS and stimulated with different secondary signals, including ATP and MSU. Supernatant IL-1 beta , IL-6 and TNF-alpha were analyzed using ELISA, n = 3 per group (A–C). Supernatants (SN) and cell extracts lysate (Lys) were analyzed using immunoblot assays (D). Cell death were detected by CCK8 asssay, n = 4 per group (E). Supernatants were collected to measure cell death by LDH release assay, n = 3 per group (F). PI staining was detected by fluorescence and the quantification of PI-positive cells is shown, n = 3 per group (G). The cleavage of GSDMD were detected using immunoblot assays (H); I, J. HEK293T cells were transfected with Flag-NLRP3, Myc-ASC, Flag-pro-caspase-1, Flag-pro-IL-1 beta plasmids as well as si-RNA of HECTD3 and stimulated with 2.5 mM ATP. Supernatants (SN) and cell extracts lysate (Lys) were analyzed using immunoblot assays (I); Supernatant IL-1 beta were analyzed using ELISA, n = 3 per group (J); K BMDMs isolated from Hectd3+/+ and Hectd3−/− mice were treated with 200 ng/mL LPS for 15 or 30 min, cell extracts were analyzed with immunoblot assays. Data are the mean ± SEM, ns (non-significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, two-tailed unpaired Student’s t test was used. Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41419-024-06473-4), licensed under a CC-BY license. Not internally tested by R&D Systems.

AP does not affect the NF-kappa B signaling pathway. (a) 293 T cells were transfected with the pGL4.32 luciferase reporter vector and treated with TNF-alpha (20 ng/mL) for 5 h with or without AP. Luciferase activity was analyzed using the Promega Bright-Glo™ Luciferase Assay System. (b) LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM). IL-1 beta in the supernatants (Sup), phospho-NF-kappa B and NF-kappa B in the soluble lysates (Lys) were analyzed using a Western blot. Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41598-024-55651-z), licensed under a CC-BY license. Not internally tested by R&D Systems.

Neutrophil recruitment contributes to BBB disruption following peripheral inflammatory stimulation.a Representative immunoblots detecting pro-IL-1 beta , IL-1 beta , and NLRP3 in brain lysates of WT mice at different time points (0, 1 ~ 6 h) after the second LPS injection. b, c Quantification of brain-infiltrating myeloid cells or neutrophils in the brain of WT mice upon PBS or repeated LPS injection at different time points by flow cytometry. (1 ~ 6 h post second LPS injection, n = 5, PBS, 4 h; 6, 3 h; 4, all other group). d Violin plot showing chemokine receptor gene expression in neutrophils of all groups. e Circle plot visualizing cell-cell communication in the CXCL signaling pathways for each experimental group. The plot displays interactions among two cell types in the PBS group and 5 cell types (Endo Endothelial cell, Peri Pericyte, Astro Astrocyte, Micro Microglia, Neutro Neutrophil) in the LPS x2 group. The width and color of arrows represent the probability of interaction. f Quantification of Evans blue depositions in the WT mouse brains upon repeated LPS injection pretreated with IgG (n = 4) or anti-Ly6G (n = 6) antibodies (50 μg, i.p.) 1 h before 1st LPS injection. g Quantification of Evans blue depositions in the WT mouse brains upon repeated LPS injection in the presence of vehicle (n = 6) or anti-CXCR2 antagonist SB225002 (n = 8) treatment (2 mg/kg, i.p.). h Quantification of Evans Blue extravasation into the brain followed by LPS injections in WT and Ccr2−/− mice. n = 5 (WT) or 3 (Ccr2−/−) mice. Error bars, s.e.m. One-way ANOVA with Dunnett post hoc test (b, c) and unpaired two-sided Student’s t test (f, g, h). Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/39814731), licensed under a CC-BY license. Not internally tested by R&D Systems.

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

Recombinant Mouse IL‑1 beta /IL‑1F2 (Catalog # <a class=

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_lightbox">

401-ML) was serially diluted and captured by Rat Anti-Mouse IL‑1 beta /IL‑1F2 Monoclonal Antibody (Catalog # MAB401) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL‑1 beta /IL‑1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-401-NA) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)" class="big_thumb" />

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

, , , ,

Applications: , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	AF-401-NAU-100UG
Alexa Fluor 405	AF-401-NAV-100UG
Alexa Fluor 488	AF-401-NAG-100UG
Alexa Fluor 532	AF-401-NAX-100UG
Alexa Fluor 594	AF-401-NAT-100UG
Alexa Fluor 647	AF-401-NAR-100UG
Alexa Fluor 700	AF-401-NAN-100UG
Alexa Fluor 750	AF-401-NAS-100UG
Alexa Fluor Plus 405	AF-401-NAAFP405
Alexa Fluor Plus 488	AF-401-NAAFP488
Alexa Fluor Plus 555	AF-401-NAAFP555
Alexa Fluor Plus 594	AF-401-NAAFP594
Alexa Fluor Plus 647	AF-401-NAAFP647
Alexa Fluor Plus 680	AF-401-NAAFP680
Allophycocyanin	AF-401-NAA
Biotin	BAF401
CoraFluor 1	AF-401-NACL1
DyLight 350	AF-401-NAD
DyLight 405	AF-401-NAE
DyLight 488	AF-401-NAK
DyLight 550	AF-401-NAL
DyLight 594	AF-401-NAM
DyLight 650	AF-401-NAW
DyLight 680	AF-401-NAY
DyLight 755	AF-401-NAZ
FITC	AF-401-NAF
HRP	AF-401-NAH
Janelia Fluor 525	AF-401-NAJF525
Janelia Fluor 549	AF-401-NAI
Janelia Fluor 646	AF-401-NAJ
Janelia Fluor 669	AF-401-NAJF669
PE	AF-401-NAP
PerCP	AF-401-NAC
mFluor Violet 450 SE	AF-401-NAMFV450
mFluor Violet 500 SE	AF-401-NAMFV500
mFluor Violet 610 SE	AF-401-NAMFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	AF-401-SP

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AF3628

CD31/PECAM-1 Antibody [Unconjugated]

(6 Reviews)

(574 Publications)

100 μg

AF3628

HAF017). A specific band was detected for CD31/PECAM-1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_lightbox">

Western blot shows lysates of bEnd.3 mouse endothelioma cell line. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (<a class=

HAF017). A specific band was detected for CD31/PECAM-1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." alt="Western blot shows lysates of bEnd.3 mouse endothelioma cell line. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for CD31/PECAM-1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_thumb" />

Simple Western shows lysates of bEnd.3 mouse endothelioma cell line, loaded at 0.5 mg/ml. A specific band was detected for CD31/PECAM‑1 at approximately 165 kDa (as indicated) using 10 µg/mL of Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628). This experiment was conducted under reducing conditions and using the 12-230kDa separation system.

AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (F0108)." class="big_lightbox">

Mouse splenocytes were stained with Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628, filled histogram) or control antibody (<a class=

AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (F0108)." class="big_thumb" />

AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (F0108)." class="big_lightbox">

Rat splenocytes were stained with Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628, filled histogram) or isotype control antibody (<a class=

AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (F0108)." class="big_thumb" />

NL003) and counterstained with DAPI (blue). Specific staining was localized to cell membrane. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_lightbox">

CD31/PECAM-1 was detected in immersion fixed bEnd.3 mouse endothelioma cell line using Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 493-conjugated Anti-Goat IgG Secondary Antibody (green; <a class=

NL003) and counterstained with DAPI (blue). Specific staining was localized to cell membrane. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_thumb" />

NL001) and counterstained with DAPI (blue). Specific staining was localized to developing endothelium. View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections." class="big_lightbox">

CD31/PECAM-1 was detected in immersion fixed frozen sections of mouse embryo (E13.5) using Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 10 µg/mL overnight at 4 °C. Tissue was stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (yellow; <a class=

NL001) and counterstained with DAPI (blue). Specific staining was localized to developing endothelium. View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections." class="big_thumb" />

VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to developing guts. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_lightbox">

CD31/PECAM-1 was detected in immersion fixed frozen sections of mouse embryo (14 d.p.c.) using Goat Anti-Human/Mouse/Rat CD31/PECAM-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 10 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (<a class=

VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to developing guts. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_thumb" />

VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to endothelial cells in vasculature." class="big_lightbox">

CD31/PECAM‑1 was detected in immersion fixed paraffin-embedded sections of rat heart using Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (<a class=

VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to endothelial cells in vasculature." class="big_thumb" />

VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to endothelial cells in chorionic villi. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_lightbox">

CD31/PECAM‑1 was detected in immersion fixed paraffin-embedded sections of Human Placenta using Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628) at 15 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # <a class=

VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to endothelial cells in chorionic villi. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents." class="big_thumb" />

AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins. " class="big_lightbox">

HUVEC cells were stained with Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628, filled histogram) or isotype control antibody (Catalog # <a class=

AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins. " class="big_thumb" />

AF3628) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for CD31/PECAM‑1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1." class="big_lightbox">

Western blot shows lysates of U937 human histiocytic lymphoma cell line. PVDF membrane was probed with 0.5 µg/ml of Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (<a class=

AF3628) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for CD31/PECAM‑1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1." class="big_thumb" />

Representative image of immunostaining corresponding to the glucose transporter protein SLC2A1 (GLUT1) in 30 week-old Ins2AKITA and WT cerebral cortex. Mural cells (ANPEP, green), glucose transporter 1 (GLUT1, red), endothelium (PECAM1, cyan). n = 2, scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30498224), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>AcSDKP suppresses TGF beta /smad signaling and EndMT through the FGFR1/FRS2 pathway. (a) HMVECs were treated with N-FGFR1 for 48 h, and the FGFR1, TGF beta R1 and TGF beta R2 protein levels were analyzed by western blot. (b) HMVECs were treated with TGF beta 2 in the presence or absence of N-FGFR1 for 15 min with or without AcSDKP preincubation. The p-smad3 and TGF beta R1 protein levels were analyzed by western blot. Densitometric analysis of the p-smad3/smad3 and TGF beta R1/ beta -actin levels (n=3) in each group was performed. (c) HMVECs were incubated with either N-FGFR1 in the presence or absence of TGF beta 2 for 48 h with or without preincubation with AcSDKP for 2 h or with N-FGFR1 in the presence or absence of TGF beta 2 for 48 h with or without 24 h of incubation with FGF2 (50 ng/ml). The CD31, SM22 alpha , FSP1 and alpha -SMA protein levels were analyzed by western blot. (d) HMVECs were transfected with FRS2 siRNA (100 nM) for 48 h with or without AcSDKP preincubation. The VE-cadherin, FSP1, vimentin, SM22 alpha and p-smad3 levels were analyzed by western blot. (e) HMVECs were treated with N-FGFR1 for 48 h or 15 min in the presence or absence of N-TGF beta (1, 2, 3) (1.0 μg/ml). The CD31, VE-cadherin, SM22 alpha , FSP1, TGF beta R1, TGF beta R2 and p-smad3 levels were analyzed by western blot Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28771231), licensed under a CC-BY license. Not internally tested by R&D Systems.

AcSDKP inhibits TGF beta /smad signaling and EndMT and restores the FGFR1 and P-MAP4K4 levels in diabetic hearts. (a) Immunofluorescence microscopy analysis of CD31/FGFR1 and CD31/P-MAP4K4 in the heart tissues from each group of mice. The scale bar is 60 μm in each panel. The CD31 and FGFR1 double-labeled cells and the CD31 and P-MAP4K4 double-labeled cells in each visual field were assessed by fluorescence microscopy and quantified. For each section, images from six different fields of view at × 400 magnification were evaluated. (b and c) Immunofluorescence microscopy analysis of CD31/ alpha -SMA, VE-cadherin /SM22 alpha and CD31/p-smad3 expression levels in the heart tissues from each group of mice. The scale bar is 60 μm in each panel. The CD31 and alpha -SMA double-labeled cells, the VE-cadherin and SM22 alpha double-labeled cells and the CD31 and p-smad3 double-labeled cells in each visual field were analyzed by fluorescence microscopy and quantified. For each section, images from six different fields of view at × 400 magnification were evaluated. Four mice from each group were analyzed. (d) Western blot analysis of the FGFR1, P-MAP4K4, TGF beta 1, TGF beta 2 and TGF beta 3 levels in cardiac tissues. A representative blot from four independent experiments was shown. The densitometric analysis of western blot data was presented (n=4). The diabetic mice are abbreviated as DM in the figure Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28771231), licensed under a CC-BY license. Not internally tested by R&D Systems.

Diminished permeability of liver sinusoids in Plvap-deficient mice.Neither by immunohistochemistry with antibodies against CD31 (A) nor by light microscopy of 1 µm semi-thin sections (B, Richardson's stain) obvious differences are detected with regards to the overall orientation and the density of liver sinusoids between 3-week-old Plvap-/- mice and wild-type littermates. Sinusoids of Plvap-deficient mice show a higher number of macrophages in their lumen (white arrows) and focal areas with accumulations of mononuclear cells in Disse's space (black arrows). Lower panels in B show higher magnifications. C, After perfusion of a wild-type animal with FITC-dextran, a strong FITC-signal (green) throughout the liver is detected. Immunolabeling with CD31 (red) suggests that FITC-dextran molecules have accumulated in the space of Disse. In contrast, in the Plvap-deficient littermate, the signal for FITC-dextran is much weaker and barely detectable. Nuclear DNA is labeled with DAPI (blue). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0115005), licensed under a CC-BY license. Not internally tested by R&D Systems.

Blood-brain barrier permeability measurements in male Ins2AKITA and WT littermate controls. (a) Representative stereomicroscope fluorescence images of brains showing 1 kDa Alexa Fluor 555 cadaverine permeability in Ins2AKITA and WT after 2 h of dye circulation (n = 2). (b) Representative confocal images of coronal sections of 1 kDa Alexa Fluor 555 cadaverine injected mouse brains. ANPEP positive mural cells, green; PECAM1 positive vasculature, white. No Alexa Fluor 555 cadaverine leakage into brain parenchyma was observed either in Ins2AKITA or WT mice (n = 2, scale bar 30 μm). (c) Quantification of 1 kDa Alexa Fluor 555 cadaverine permeability in 26.5–32 week-old Ins2AKITA and WT mice after 2 h circulation (n = > 8, 3 independent experiments). y-axis shows the fold change in relative fluorescence units (RFU) per gram of brain tissue in relation to WT. (d) Quantification of 1 kDa Alexa Fluor 488 cadaverine permeability in 38 week-old Ins2AKITA and WT mice after 2 h circulation (n = 3). y-axis shows the fold change in relative fluorescence units (RFU) per gram of brain tissue in relation to WT. (e) Evans Blue dye permeability in 30 week-old Ins2AKITA and WT littermate control mice after overnight circulation (n = > 2). y-axis shows optical density (OD) at 620 nm per gram of tissue. PdgfbRet/Ret served as positive control for tracer leakage into the brain parenchyma. n.s., not significant, student’s t test. Data is presented as mean ± SEM. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30498224), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>CL and low temperature lead to WAT browning, angiogenesis, and an increased number of myofibroblast-like cells. a, b Histological analysis of adipocyte morphology (H&E), adipocytes (PERI), mitochondria (COX4), uncoupling protein 1 (UCP1), blood vessels (CD31), and myofibroblast-like cells ( alpha SMA) in a 5-day CL-316243-treated scWAT and visWAT compared to vehicle-treated control. b Two-week 4 °C-treated scWAT and visWAT compared to 30 °C control. Double-headed arrows mark adipocyte diameter. Arrows point to respective positive signals. c–l Quantifications of adipocyte size and positive signals per field of COX4, UCP1, CD31, and alpha SMA of CL-316243- and vehicle-, and 30 °C- and 4 °C- treated scWATs and visWATs (>30 adipocytes per field; n = 10 random fields; n = 5 mice per group). PERI, perilipin; COX4, mitochondrial complex 4; UCP1, uncoupling protein 1. Scale bars, 100 μm. NS, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t-test. Data presented as mean ± s.e.m. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-017-02158-z), licensed under a CC-BY license. Not internally tested by R&D Systems.

AB-108-C, open histogram) followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins. " class="big_lightbox">

Whole blood granulocytes were stained with Goat Anti-Human/Mouse/Rat CD31/PECAM‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3628, filled histogram) or isotype control antibody (Catalog # <a class=

AB-108-C, open histogram) followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins. " class="big_thumb" />

Neutrophil depletion reduces BBB breakdown and increases neovascularization after stroke. f, g Representative confocal images (f) and quantitative analysis of IgG extravascular deposits (g) in the peri-infarct cortex at 14 days in sham-operated mice and mice treated with control antibody or anti-Ly6G antibody (n = 6). One-way ANOVA test was applied with *P < 0.0001 (Sham vs. Isotype), *P = 0.0041 (Isotype vs. Anti-Ly6G). Bar = 15 μm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Increased vascular remodeling by delayed inhibition of NET formation.a–d Representative confocal images (a, c) and quantitative analysis of IgG extravascular deposits (b, d) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and treated with either anti-Ly6G antibody, control antibody, DNase 1, or vehicle starting at 7 days (n = 6), unpaired two-tailed Student’s t-test was applied with *P = 0.0392 (b), *P = 0.0384 (d). Bar = 10 μm. e–l Representative confocal images (e, g) of CD31-positive microvessels and in-vivo multiphoton microscopy images of perfused cortical capillaries with intravenously injected FITC-dextran (i, k) in the peri-infarct cortex at 14 days in mice treated with either anti-Ly6G antibody, control antibody, DNase 1, or vehicle. Bar = 40 μm (e, g) and 100 µm (i, k). Quantification of microvascular density (f, h) and perfused capillary length (j, l) for each group (n = 6), unpaired two-tailed Student’s t-test was applied with *P = 0.00378 (f), *P = 0.0364 (h), *P = 0.0026 (j), *P = 0.0006 (l). Data are presented as mean ± SD. Source data underlying graph b, d, f, h, j, and l are provided as a Source Data file. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Neutrophil depletion reduces BBB breakdown and increases neovascularization after stroke. h, j Representative confocal images (h) of CD31-positive microvessels and in-vivo multiphoton microscopy images of perfused cortical capillaries with intravenously injected FITC-dextran (MW = 2000,000 Da) (j) in the peri-infarct cortex at 14 days in mice treated with control antibody or anti-Ly6G antibody, compared with sham-operated mice. Bar = 50 μm (e) and 100 µm (g) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Neutrophils accumulate in the brain during all stages of ischemic stroke. f Representative confocal images of Ly6G-labeled neutrophils (green) and CD31-positive microvessels (white) in the peri-infarct cortex of mice at 3 days. Nuclei were visualized with Hoechst. Neutrophils were observed within brain vessels and migrated into the parenchyma. Bar = 40 μm. Independent experiments are repeated at least three times. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32427863), licensed under a CC-BY license. Not internally tested by R&D Systems.

Microvascular and tumor cell biology at the tumor rim in MDA231Br-GFP brain metastases. Representative histologic sections of rat brains, intrastriatally injected with MDA231Br-GFP tumor cells, from the tumor core, tumor rim, and the contralateral striatum, with corresponding box and whisker plots of marker expression in each region. Data shown as median ± interquartile range. Sections were immunohistochemically stained (brown) for tumor cell marker vimentin (A), endothelial marker CD31 (B), cell adhesion molecule VCAM-1 (C), cell proliferation marker Ki67 (D), and two stemness markers: SOX2 (E) and nestin (F). Scale bar = 100 μm. *, P < 0.05; ***, P < 0.001; n = 8; post hoc Bonferroni multiple comparison test for tumor cell density and microvessel density, and post hoc Dunn test for VCAM-1 expression. Tumor core was delineated from the infiltrative border as indicated by the blue and red lines, respectively. Expression of Ki67, nestin and SOX2 has been normalized to tumor area. *, P < 0.05; ***, P < 0.001; matched Wilcoxon test. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35312755), licensed under a CC-BY license. Not internally tested by R&D Systems.

Microvascular and tumor cell biology at the tumor rim in U87MG glioblastoma. Representative histologic sections of rat brains, intrastriatally injected with U87MG tumor cells, from the tumor core, tumor rim, and the contralateral striatum, with corresponding box and whisker plots of marker expression in each region. Data shown as median ± interquartile range. Sections were immunohistochemically stained (brown) for tumor cell marker vimentin (A), endothelial marker CD31 (B), cell adhesion molecule VCAM-1 (C), cell proliferation marker Ki67 (D), and two stemness markers: SOX2 (E) and nestin (F). Scale bar = 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 5; post hoc Bonferroni multiple comparison test for tumor cell density and microvessel density, and post hoc Dunn test for VCAM-1 expression. Tumor core was delineated from the infiltrative border as indicated by the blue and red lines, respectively. Expression of Ki67, nestin, and SOX2 has been normalized to tumor area. **, P < 0.01; matched Wilcoxon test. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35312755), licensed under a CC-BY license. Not internally tested by R&D Systems.

Immunohistochemistry (paraffin-embedded) of Native Adult Rat lung tissue stained for Abca3 (red), CD31/PECAM-1 (green), and Dclk1 (cyan). Image from a verified customer review.

IF staining of human lung endothelial cells with cd31/pecam1. Image from a verified customer review.

Immunostaining analysis of angiogenic markers. (a) Immunohistochemistry (IHC) staining and semi‐quantitative analysis of CD31 in regenerated Achilles tendon of control, suture repair (SR), sodium alginate (SA), and bioactive glass (BG)/SA groups on Days 3, 7, 14, and 28 post‐surgery. Scale bar = 100 μm. T, tendon tissue; G, granulation tissue. Black arrows denote positive signals. Results of statistical analysis are presented as means ± SD; (n = 3; *p < 0.05). (b) Immunofluorescence (IF) staining of CD31 and alpha ‐SMA in regenerated Achilles tendon of SR, SA, and BG/SA groups on Days 7, 14, and 28 post‐surgery. Scale bar = 50 μm; Scale bar (zoom) = 20 μm Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36684098), licensed under a CC-BY license. Not internally tested by R&D Systems.

FGF-2 expression in NPC correlates with tumor vasculature.A Transcriptomic expression levels of angiogenic factors, including VEGFA, FGF2, PDGFB, EGF, ANGPT1, EPO in human KIRC tissues, COAD tissues, NPC tissues, STAD tissues, PAAD tissues, LUAD tissues, BRCA tissues, SKCM tissues, and their adjacent healthy tissues. The red line indicates the highest expression of FGF2 in AAD-resistant NPC and the lowest expression of FGF in AAD-sensitive CRC. B Transcriptomic expression levels of FGF2 in NPC tissues, COAD tissues, and their adjacent healthy tissues (sample number: control-NPC/NPC/control-COAD/COAD = 10/31/41/290). Data were extracted from datasets GSE12452 and TCGA. C Human rhinitis tissues and NPC tissues were collected and detected for histology (H&E), FGF-2, and CD31 expression levels. Scale bar in upper panel = 500 μm, scale bar in lower three panels = 50 μm. Quantification of FGF-2+ or CD31+ signals (n = 8 random fields per group). D QPCR quantification of FGF2 and CD31 expression in freshly collected rhinitis tissues and NPC tissues (rhinitis tissue, n = 5 samples; NPC tissue, n = 6 samples). E Correlation of FGF2 and CD31 transcriptomic expression levels of human NPCs and control rhinitis tissues (Rhinitis tissue, n = 5 samples; NPC tissue, n = 6 samples). *P < 0.05; ***P < 0.001. NS not significant. Data presented as mean ± SD. KIRC kidney renal clear cell carcinoma, COAD colon adenocarcinoma, STAD stomach adenocarcinoma, PAAD pancreatic adenocarcinoma, LUAD lung adenocarcinoma, BRCA breast invasive carcinoma, SKCM skin cutaneous melanoma. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35985991), licensed under a CC-BY license. Not internally tested by R&D Systems.

Increase in renal fibrosis in aging kidneys of Atg7flox/flox;Tie2-Cre+ mice. (A) Representative immunoblots and densitometry for the expression of Atg7. (B) Relative protein level of Atg7 (%). (C) Representative images of H&E staining from wild-type and Atg7flox/flox;Tie2-Cre+ mice. Scale bars, 100 μm. (D) Quantification of glomeruli diameters (µm). (E) Representative 3,3′-diaminobenzidine (DAB) staining for CD31. Scale bars, 50 μm. (F) Quantification of capillary lumen diameters (µm). (G) Representative immunoblots and densitometry for the expression of CD31. (H) Relative protein levels of CD31 (%). (I) Masson’s trichrome staining from wild-type and Atg7flox/flox;Tie2-Cre mice, showing the cortex and medulla with increased extracellular matrix deposition in the aging Atg7flox/flox;Tie2-Cre+ mouse. (J) Quantification of tubulointerstitial fibrosis (%). (K) Representative DAB staining for TGF-beta . (L) Quantification of TGF-beta positive areas (%). (M) Representative immunoblots and densitometry for expression of TGF-beta . (N) Relative protein level of TGF-beta (%). (O) Representative DAB staining for alpha -SMA. (P) Quantification of alpha -SMA-positive areas (%). (Q) Representative immunoblots and densitometry for expression of alpha -SMA. (R) Relative protein levels of alpha -SMA (%). Scale bars, 50 μm for cortex and 100 μm for medulla. Values are means ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, ns: not significant. Image collected and cropped by CiteAb from the following open publication (//www.mdpi.com/2076-3921/13/8/886), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Hu, Mu, Rt

Species Glossary

Reactivity: Hu, Mu, Rt

Applications:

, , , , ,

Applications: , , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	AF3628U-100UG
Alexa Fluor 405	AF3628V-100UG
Alexa Fluor 488	FAB3628G-025
Alexa Fluor 488	FAB3628G-100
Alexa Fluor 532	AF3628X-100UG
Alexa Fluor 594	AF3628T-100UG
Alexa Fluor 647	AF3628R-100UG
Alexa Fluor 700	AF3628N-100UG
Alexa Fluor 750	AF3628S-100UG
Alexa Fluor Plus 405	AF3628AFP405-100UG
Alexa Fluor Plus 488	AF3628AFP488-100UG
Alexa Fluor Plus 555	AF3628AFP555-100UG
Alexa Fluor Plus 594	AF3628AFP594-100UG
Alexa Fluor Plus 647	AF3628AFP647-100UG
Alexa Fluor Plus 680	AF3628AFP680-100UG
Allophycocyanin	FAB3628A-100
Allophycocyanin	FAB3628A-025
Biotin	BAF3628
CoraFluor 1	AF3628CL1
DyLight 350	AF3628D
DyLight 405	AF3628E
DyLight 488	AF3628K
DyLight 550	AF3628L
DyLight 594	AF3628M
DyLight 650	AF3628W
DyLight 680	AF3628Y
DyLight 755	AF3628Z
FITC	AF3628F
HRP	AF3628H
Janelia Fluor 525	AF3628JF525
Janelia Fluor 549	AF3628I
Janelia Fluor 646	AF3628J
Janelia Fluor 669	AF3628JF669
PerCP	AF3628C
Phycoerythrin	FAB3628P-100
Phycoerythrin	FAB3628P-025
mFluor Violet 450 SE	AF3628MFV450
mFluor Violet 500 SE	AF3628MFV500
mFluor Violet 610 SE	AF3628MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	AF3628-SP
Carrier Free	FAB3628P-100
Carrier Free	FAB3628P-025
Carrier Free	FAB3628G-100
Carrier Free	FAB3628G-025
Carrier Free	FAB3628A-100
Carrier Free	FAB3628A-025

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NB100-479

HIF-1 alpha Antibody - BSA Free

(28 Reviews)

(546 Publications)

0.1 ml

NB100-479

ChIP-qPCR assay of HIF-1 binding to hypoxia response element (HRE) of the PKM2 gene in human MDA-MB-231 breast cancer cells treated with vehicle or DMOG (HIF inducer) for 8 hours. Negative control: immunoprecipitation was performed with IgG. Image courtesy of Gregg Semenza, Johns Hopkins University School of Medicine.

HIF-1-alpha-Antibody-Immunohistochemistry-NB100-479-img0030.jpg

Staining in normal mouse kidney showing (A) staining with HIF-1 alpha antibody and (B) blocking peptide used at 10-fold excess concentration. Images provided by Dr Yves Heremans. Please see the product review of NBC1-18422 for additional details.

<b>Biological Strategies Validation. </b>Analysis of HIF-1 alpha in mouse hepatocytes (left lane: normoxic and right lane: hypoxic) using HIF-1 alpha antibody. Western blot image submitted by a verified customer review.

Analysis in paraffin section of mouse skin wound using NB100-479 at 1:6000. Image courtesy of product review by Ian Darby of RMIT University.

<b>Biological Strategies Validation. </b>HIF-1-alpha-Antibody-Immunocytochemistry-Immunofluorescence-NB100-479-img0027.jpg

<b>Biological Strategies Validation. </b>Analysis in A. PC12 (rat) cells, 20% oxygen and B. PC12 (rat) cells, 1% oxygen.

<b>Biological Strategies Validation. </b>Analysis in a hypoxic sample. Lane 1: CoCl2 treated Cos-7 nuclear extract (50 ug, hypoxic). Lane 2: Untreated Cos-7 nuclear extract (50 ug, normoxic). 10 second ECL exposure.

U251 cells exposed to 4 hour hypoxia (0.5% oxygen) incubation. Western blot image submitted by a verified customer review.

Detection of HIF-1 Alpha (green) in RCC4 cells using NB100-479. Nuclei (Blue) were counterstained using Hoechst 33258.

Analysis of HIF-1 alpha in human kidney tumor.

HIF-1-alpha-Antibody-Immunohistochemistry-Paraffin-NB100-479-img0028.jpg

HIF-1-alpha-Antibody-Immunohistochemistry-NB100-479-img0026.jpg

Simple Western lane view shows lysates of MCF +/- DMOG, BioSpherix MSCs in hypoxic conditions, and BioSpherix MSCs in normoxic conditions loaded at 0.5 mg/ml. A band was detected for HIF-1 alpha at approximately 116 kDa (as indicated) using NB100-479 (1:100 dilution) followed by Anti-Rabbit Secondary Antibody (042-206, ProteinSimple). This experiment was conducted under standard assay conditions, and using the 12-230 kDa separation module (SW-W004). Non-specific interaction with the 230 kDa Simple Western standard seen with this antibody. Image from an internal validation.

<b>Genetic Strategies Validation. </b>Cofilin phosphorylation is HIF-1 alpha dependent.Wt, shC, and the HIF-1 alpha knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O2) or hypoxia (1% O2). Cell extracts were analysed by Western blots. Note that p-cofilin levels are reduced in c2 and c2 cells compared to wt and shC cells at 20% O2 and 1% O2.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxic markers accumulate in miR-29-sponge Zebrafish hearts. (A) Representative western blot analysis of hypoxia inducible factor 1 alpha (HIF1 alpha ) expression in Wild Type & miR-29-sponge Zebrafish heart. In each condition, alpha -tubulin was used as loading control. Three independent experiments were performed. Full-length blot is presented in Supplementary Figure 6.(B) qRT-PCR mRNA analysis of hypoxia associated genes: erythropoietin alpha (epoa); hexokinase2 (hk2); heme oxygenase1a (hmox1a); lactate dehydrogenase A (ldha); cyclin-dependent kinase inhibitor 1B (p27) in Wild Type (black circles; n = 4) & miR-29-sponge (gray squares; n = 4) Zebrafish hearts expressed as fold-change versus Wild Type samples. *p < 0.05 Vs Wild Type. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-16829-w), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-479] - (a,b) Immunofluorescence staining of (a) HIF-1 alpha (green), the macrophage marker F4/80 (red) & DAPI nuclear stain (blue), & (b) lactate dehydrogenase (LDH, green), F4/80 (red) & DAPI (blue) in WAT of lean & obese mice. Colocalization is shown in the merged image (arrows). Scale bar = 100 µm. (c,d) Relative mRNA expression of (c) Hif1a & Hif2a, & the HIF target genes (d) Vegfa & Glut1 in ATM isolated from lean & obese mice (n = 3). (e) Levels of succinate in ATM of lean & obese mice (n = 6). (f) Relative mRNA levels of the glutamate transporter Slc3a2 in ATM from lean & obese (n = 3). Data is expressed as mean ± s.e.m. *p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32221369), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Increased expression of HIF alpha and/or Twist in A549 & H441 cells induced by the inhibition of PIMT & Thapsigargin(A) Immunoblotting of Slug, ZEB1, Snail1, Twist, & HIF1 alpha in A549 sh-PIMT & sh-control cells. (B, C) Immunoblotting & relative intensity of HIF1 alpha in A549 cells treated with Tg. (D) Immunoblotting of Slug, ZEB1, Snail1, Twist, & HIF1 alpha in si-control cells & si-PIMT H441 cells. (E, F) Immunoblotting & relative intensity of HIF1 alpha in H441 cells treated with Tg. #1 & #2 indicates si-RNA of J-010000-05-0002 & J-010000-07-0002, respectively. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.24324), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-479] - Histology & immunofluorescence in rat corneal sections. a Hematoxylin & Eosin (H&E), b VEGF-A (green); c CCL2 (green); d TNF-alpha (green); e CXCL5 (green); f CD45 (green) & g HIF-1 alpha (green) staining in rat cornea tissue. Nuclear counterstaining by DAPI (blue) in fluorescent images Image collected & cropped by CiteAb from the following publication (//link.springer.com/10.1007/s10456-018-9594-9), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody - BSA Free [NB100-479] - White-jaded spleen & muscle in another NOD/SCID mouse model. One-week-old NOD/SCID mice were i.p. infected with EV-A71, & scarified after disease manifestation. aUpper panel: An atrophic & discolored spleen was identified in EV-A71-infected mice. Viral protein (VP1) & HIF1A were both expressed in the infected spleen. Lower panel: A saline control showed a normal-sized spleen with no VP1 & less expression of HIF1A. b Both HIF1A & VEGFA were strongly expressed in the whitened muscles infected with EV-A71, but not detectable in the saline control Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31711481), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - The Neutralizing antibody against nucleolin eliminate HDGF-stimulated AKT/HIF-1 alpha /NF-kappa B/VEGF signaling in SCC4 oral cancer cells. a-d SCC4 cells were treated with recombinant HDGF protein (100 ng/ml) in the presence of anti-NCL or anti-IgG antibody (5 μg/ml) for 24 h before total protein extraction. Cell lysates were subjected to Western blotting with the indicated antibodies. beta -actin was used as an internal control for loading & transfer. Data were mean of three experiments. *, P < 0.05; **, P < 0.01; ns, not statistically significant Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31711427), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hif1 alpha expression in neonatal & adult ovary.(A) Section of P5 ovary showing MVH expression in all oocytes & Hif1 alpha expression only in small oocytes (primary follicles; top). Negative control images without primary antibodies (bottom). Scale bar: 50μm. (B) Section of adult ovary showing absence of Hif1 alpha expression in primary follicles detected with MVH staining (top). Negative control without primary antibody (bottom). Scale bar: 50μm. (C) Section of adult ovary showing Hif1 alpha expression in mature primary oocyte (arrow). Negative control without primary antibody (bottom). Scale bar: 100μm. (D) Image of a Metaphase II oocyte from superovulated three-month-old female showing Hif1 alpha expression. Scale bar: 50μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27148974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of alpha -MSH on HIF-1 alpha expression & activity in B16-F10 melanoma cells during hypoxia.a, b alpha -MSH increased HIF-1 alpha mRNA & protein expression levels during hypoxia. c Cells were transfected with luciferase reporter driven by a promoter region containing HIF-1 alpha -specific binding sites before alpha -MSH treatment at the indicated time & concentration. The luciferase activities were measured immediately & expressed as means ± SD from triplicate experiments. d Relative mRNA expression levels were analyzed by real-time PCR. Data are expressed as fold change compared with control (means ± SD of triplicate experiments). *p < 0.05, **p < 0.01. alpha -MSH elevated the Bcl-2 family gene expression levels by HIF-1 alpha activation Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30062060), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-479] - (a) Immunofluorescence staining for HIF-1 alpha (red), hypoxia probe pimonidazole (green) & DAPI nuclei (blue) in WAT of obese mice. Boxed region in the merged image shows HIF-1 alpha -positive cells in crown-like structures that are Pimo− (arrows). (b) HIF-1 alpha protein levels in BMDM treated with BSA (control) or BSA-conjugated palmitate (Palm) for 6, 12 & 24 hours. Tubulin is shown as an internal loading control. (c) Fold change in mRNA expression of Hif1a, Vegfa & Il1b in BMDM treated with palmitate or BSA. (d) Relative mRNA expression in BMDM treated as indicated in the presence or absence of a HIF-1 alpha inhibitor. Data are representative of 3 independent experiments & are expressed as mean ± s.e.m. *p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32221369), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hif1 alpha mediates Glut1 induction downstream of Bmp-mTORC1 signaling. a–c Western blot (a, c) or RT-qPCR (b) analyses in primary chondrocytes after 24 h of BMP2 treatment with or without mTOR inhibitors. Rapa: rapamycin. d Western blot analyses in primary chondrocytes in response to BMP2 with or without the Hif1 alpha inhibitors for 24 h. Quantification of all western blots denotes average fold change over vehicle control after normalization to beta -actin (±SD, n = 3). *p < 0.05, n = 3, error bar indicates SD, two-tailed Student’s t-test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30446646), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Tamoxifen administration-induced PHD2 deletion in skeletal muscle in Phd2f/f/Rosa26CreERT2 mice. a. PHD1, 2, & 3 deletion efficiency in the gastrocnemius muscles of tamoxifen-treated Phd2f/f/Rosa26CreERT2 mice was determined using qRT-PCR. Relative gene expression was determined using gastrocnemius muscle tissue cDNA (n = 3–4 mice per group). b. Anti-PHD2 & PHD3 Western blotting of gastrocnemius & soleus muscles at 6 weeks after tamoxifen administration. c. The expression of HIF-1 alpha in gastrocnemius & soleus at 5 weeks after tamoxifen administration. d. The level of hif-1 alpha mRNA in gastrocnemius at 5 weeks after tamoxifen administration. *p < 0.05; **p < 0.01 compared to control. Values are means ± SEM Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26949511), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - FASN Regulates HIF-alpha Protein Stability & HIF Target Gene Expression. (A) FASN siRNA downregulates HIF-alpha protein levels. PREC, HeLa & HCT116 cells treated with FASN (+) or control (−) siRNA & harvested after 72 h. Cells exposed to hypoxia (1% oxygen) or maintained in normoxia (21% oxygen) 4 h before harvesting & subjecting WCEs to WB analysis. Unmodified (250 kDa) & modified (larger) FASN bands indicated by large arrowheads, REDD1 by a small arrowhead, & HIF-1 alpha & HIF-2 alpha by arrows. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28775317), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - FASN Regulates HIF-alpha Protein Stability & HIF Target Gene Expression. (B) Downregulation of HIF-1 alpha & HIF-2 alpha in HCT116 cell clones carrying the FASN gene mutagenized using CRISPR-Cas9 (See Supplementary Fig. S3 for genomic DNA information). Protein samples prepared in normoxia (left column) or hypoxia (right column) for WB analysis. Asterisk indicates a non-specific band. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28775317), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of silencing Cx43 on the expression of HIF-1 alpha in astrocytes.Astrocytes were transfected with NT-siRNA or with Cx43-siRNA. At the indicated times the expression of HIF-1 alpha & Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1 alpha , Cx43 & GAPDH showing that the decrease in Cx43 expression was concomitant with HIF-1 alpha up-regulation. B) HIF-1 alpha quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the NT-siRNA condition at time 0. ***p<0.001 versus the corresponding NT-siRNA values. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0032448), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - HDGF triggered AKT/HIF-1 alpha /NF-kappa B signaling in SCC4 oral cancer cells. a-d Cells were treated with recombinant HDGF (1–100 ng/ml) for 24 h & then harvested for total protein extraction. The cell lysates were separated by SDS-PAGE & detected by Western blotting with the indicated primary antibodies. beta -actin was used as an internal control for loading & transfer. Data were mean of three experiments. *, P < 0.05; **, P < 0.01; ns, not statistically significant Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31711427), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Cofilin phosphorylation is HIF-1 alpha dependent.Wt, shC, & the HIF-1 alpha knock down cell clones c1 & c2 cells were lysed after 24 hrs of normoxia (20% O2) or hypoxia (1% O2). Cell extracts were analysed by Western blots. Note that p-cofilin levels are reduced in c2 & c2 cells compared to wt & shC cells at 20% O2 & 1% O2. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0069128), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Genotyping of transgenes.PCR genotyping was performed for all four transgenes as described in materials & methods. Sizes of the amplified products obtained are: 240 bp for Hif-1 alpha (wild type); 274 bp for Hif-1 alpha flox/flox; 410 bp for Hif-2 alpha (wild type); 444bp for Hif-2 alpha flox/flox; 370 bpfor Cre transgene; 350 bpfor rtTA transgene. One representative sample was genotyped for the four transgenes from each of three generated mouse strains: SPC-rtTA-/tg/(tetO)7-Cre-/tg/Hif-1 alpha fl/fl (Lane 1), SPC-rtTA-/tg/(tetO)7-Cre-/tg/Hif-2 alpha fl/fl mouse (Lane 2), & SPC-rtTA-/tg/(tetO)7-Cre-/tg/Hif-1 alpha /2 alpha fl/fl (Lane 3)). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0139270), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of 24 h UO on renal HIF-1 alpha /2 alpha expression.WT mice were exposed to either sham-operation or left UO, which continued for 24 hours, & then was released. The left kidneys were harvested immediately (d0), 2, 4 or 7 days after release of obstruction (n = 4 at each time point). Immunoblot analyses of HIF-1 alpha & HIF-2 alpha in left kidneys were then performed & co-detection of TBP was performed to assess equal loading. HIF protein bands were quantified & normalized to TBP. Data were expressed as mean ± SD, & the mean value obtained from non-operated control mice was arbitrarily defined as 1. *, p<0.05 versus sham-operated controls; **, p<0.05 versus all the other groups. C, non-operated controls; S, sham-operated controls. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/22295069), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hif1 alpha expression in 15.5dpc germ cells.(A) Sections of male (top) & female (bottom) gonads from 15.5 dpc Oct4-GFP embryos showing Oct4-GFP & Hif1 alpha expressions in germ cells. Scale bars: 50μm (male) & 20μm (female). (B) FACS-sorted male (top) & female (bottom) 15.5dpc germ cells showing Oct4-GFP & Hif1 alpha expressions. Scale bars: 100μm (male) & 50μm (female). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27148974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of ET-1 on glucose uptake in HIF-1 alpha -silenced astrocytes.Astrocytes were transfected with NT-siRNA or with HIF-1 alpha -siRNA. After 48 h, astrocytes were incubated in the absence (control, C) or presence of 0.1 µM ET-1 for 24 h. A) HIF-1 alpha , Hx-1, GLUT-3, Hx-2, GLUT-1 & GAPDH Western blots & quantification. The results are the means ± SEM of at least three independent experiments & they are expressed as percentages of the level found in the control NT-siRNA. B) Glucose uptake expressed as pmol of 2-deoxyglucose (2-DG) taken up per hour & per milligram of protein. The results show that the down-regulation of HIF-1 alpha levels promoted by HIF-1 alpha -siRNA decreased the rate of glucose uptake & the expression of GLUT-1, GLUT-3, Hx-1 & Hx-2, both in the control & in the ET-1 treated astrocytes. ***p<0.001, **p<0.01 & *p<0.05 versus the corresponding controls (C); ###p<0.001, ##p<0.01 & #p<0.05 versus the corresponding NT-siRNA. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0032448), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of ET-1 & Cx43 on HIF-1 alpha expression & glucose uptake when c-Src is inhibited.Astrocytes were preincubated with 100 ng/µL PP2 (c-Src inhibitor) or 100 ng/µL PP3 (inactive analogue) for 1 h. Then, cells were incubated in the absence (control) or presence of 0.1 µM ET-1 for 24 h. A) HIF-1 alpha Western blot & quantification. The results are expressed as percentages of the level found in the controls treated with PP3 & they show that the inhibitor of c-Src PP2 prevented the up-regulation of HIF-1 alpha promoted by ET-1. ***p<0.001 versus the absence of ET-1. B) Glucose uptake expressed as pmol of 2-deoxyglucose taken up per hour & per milligram of protein. The results show that the inhibitor of c-Src PP2 prevented the increase in the rate of glucose uptake promoted by ET-1. ***p<0.001 versus the absence of ET-1. C) Astrocytes were preincubated with 100 ng/µL PP2 or 100 ng/µL PP3 for 1 h. Then, cells were transfected with NT-siRNA or with Cx43-siRNA & after 48 h HIF-1 alpha was analysed by Western blot. The results are expressed as percentages of the level found in the PP3 NT-siRNA & they show that the inhibitor of c-Src PP2 prevented the up-regulation of HIF-1 alpha promoted by silencing Cx43. ***p<0.001 versus the corresponding NT-siRNA. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0032448), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia reduces inflammatory processes & induces autophagy in Il-10−/− mice. a, b, c, d, e, f & g WT, Nrlp3−/−,Il-10−/−, & Il-10−/−Nrlp3−/−double knockout mice were subjected to normoxia (N, 21% O2) or hypoxia (H, 8% O2). After 18 h, mice were killed & colon biopsies were collected. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test. Results represent mean + s.e.m., WT mice under normoxia: n = 5, WT mice under hypoxia: n = 5; Nrlp3−/− mice under normoxia: n = 4; Nrlp3−/− mice under hypoxia: n = 5; Il-10−/− mice under hypoxia: n = 4; Il-10−/− mice under hypoxia: n = 4; Il-10−/−Nrlp3−/− mice under normoxia: n = 7; Il-10−/−Nrlp3−/− mice under hypoxia: n = 9, *P < 0.05; **P < 0.01; ***P < 0.001. h Total protein was isolated & western blot performed. LC3-I & LC3-II bands were quantified, & autophagy was measured by variations in the ratio of LC3-II/LC3-I & the total amount of LC3 (LC3-I plus LC3-II) relative to beta -actin Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28740109), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia reduces inflammatory processes & induces autophagy in the DSS mouse model of colitis. a, b, c, d, e, f & g WT & Nrlp3−/− mice were administered with DSS or DSS-free water & subjected to normoxia (N, 21% O2) or hypoxia (H, 8% O2). After 18 h, mice were killed & colon biopsies were collected. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test. Results represent mean + s.e.m., WT mice under normoxia: n = 5, WT mice under normoxia: n = 5, DSS-treated WT mice under normoxia: n = 6; Nrlp3−/− mice under normoxia: n = 5; DSS-treated Nrlp3−/− mice under normoxia: n = 6; WT mice under hypoxia: n = 4, DSS-treated WT mice under hypoxia: n = 5; Nrlp3−/− mice under hypoxia: n = 3; DSS-treated Nrlp3−/− mice under hypoxia: n = 3, *P < 0.05; **P < 0.01; ***P < 0.001. h Total protein was isolated & western blot performed. LC3-I & LC3-II bands were quantified, & autophagy was measured by variations in the ratio of LC3-II/LC3-I & the total amount of LC3 (LC3-I plus LC3-II) relative to beta -actin Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28740109), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of HIF-1 alpha silencing on alpha -MSH-induced apoptosis in B16-F10 melanoma cells during hypoxia.a Cells were transfected with shLacZ or shHIF-1 alpha plasmids for 48 h before harvest. HIF-1 alpha shRNA reduced the basal HIF-1 alpha mRNA & protein expression levels. b, c Relative mRNA expression levels were analyzed by real-time PCR. Data are expressed as fold change compared with control (means ± SD of triplicate experiments). d Cell lysates were analyzed by immunoblot using the indicated antibodies. beta -Actin was used as an internal control for loading & transfer. e The population of apoptotic cells was analyzed by flow cytometry & qualified as mean ± SD from triplicate experiments. *p < 0.05, **p < 0.01. Knockdown of HIF-1 alpha diminished alpha -MSH-induced apoptosis in B16-F10 melanoma cells during hypoxia Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30062060), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - FASN & CAD Binding Specificity of pVHL Mutants Derived from VHL Patients. (A) Schematic representation of pVHL domains. Dotted lines indicate the approximate positions of the HIF-1 alpha & Elongin C binding domains. The alpha & beta structural domains are also indicated. (B) Effects of pVHL mutations on interactions with E3 ligase components, HIF-alpha & other interacting proteins including FASN. pVHL mutants were stably expressed by lentiviral vectors in HCT116 cells, & WCEs were tested in precipitation assays. Note that the mutant pVHL-W88S almost completely lost the ability to bind to HIF-1 alpha & HIF-2 alpha , whereas pVHL-Y112H retains this ability almost completely. However, both pVHL-W88S & pVHL-Y112H show significantly reduced binding to FASN & CAD, as indicated by small & large asterisks, respectively. Arrows indicate HIF-1 alpha & HIF-2 alpha bands. Long arrows & arrowheads indicate unmodified & modified forms of FASN, respectively. Protein samples prepared from hypoxia (1% oxygen)-exposed cells were added as positive control in left-end extra lanes. (C) The same samples as used in B were analyzed by silver staining. The E3 ligase components appear as major bands as indicated. (D) Subcellular localization of stably expressed pVHL mutants in 786-O. pVHL-Y112H & pVHL-W88S showed preferential nuclear localization, whereas pVHL-WT & pVHL-H115Q which both strongly bind to FASN & CAD, showed cytoplasmic localization. Cells showing clear nuclear (C < N) or cytoplasmic (C > N) localization in three microscopic viewing areas were counted as shown in the lower panel. The total counted cell numbers ranged from 35 to 64. The experiments were repeated more than twice, except for the cell localization count, & the results were reproduced. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28775317), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia signaling & cytotoxicity to CdCl2. (a) Wild-type (WT) cells were left untreated (Ctrl) or exposed to 150 μM CoCl2 (Co2+), 1, 5 or 10 μM CdCl2 (Cd2+) or 150 μM CoCl2 & 10 μM CdCl2 for 24 hours. HIF1 alpha −/− cell extract treated with 150 μM CoCl2 was used as a negative control. Nuclear protein was extracted & separated by SDS-PAGE, transferred to nitrocellulose membrane & probed with a HIF1 alpha (upper panel) or beta -actin (lower panel) specific antibody. The bands observed in the cadmium only WT cells are nonspecific as they are also observed in the HIF1 alpha −/− cells. (b) BNip3 mRNA expression levels were analyzed by qRT-PCR in wild type (WT, white bars) & HIF1 alpha −/− cells (black bars). Cells were left untreated (0), or exposed to 5 μM CdCl2 (Cd2+) or 150 μM CoCl2 (Co2+) for 24 hours. Each value was normalized to the control level in the corresponding cell line. *P < 0.05 compared to the corresponding controls, n = 4. (c) BNIP3 protein levels were determined in wild type & HIF1 alpha −/− cells after treatment with 150 μM CoCl2 (Co2+) or 5 μM CdCl2 (Cd2+) for 24 hours using a BNIP3 specific antibody & beta -actin was used as a loading control (lower Panel). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21811500), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody - BSA Free [NB100-479] - FIH exits the nucleus via a Leptomycin B-sensitive exportin1-dependent pathway. (A) Immunofluorescence staining of FIH (green) & HIF1 alpha (red) in MCF7 cells after the indicated hypoxia (0.5% O2) & re-oxygenation treatments. TO-PRO-3 (blue) was used to stain nuclei. (B) Total cell lysates from U2OS cells were immunoprecipitated with an anti-exportin1 antibody or control IgG. FIH, exportin1 & beta -tubulin levels are indicated. (C) Immunofluorescence staining of FIH (green) in FIH-null mouse embryonic fibroblasts (MEFs) transfected with HA-FIH 1–349 or HA-FIH delta NES followed by normoxia, hypoxia (1% O2, 3 h) or 3 h of hypoxia followed by re-oxygenation for 1 h. TO-PRO-3 (blue) was used to stain nuclei. Arrows indicate nuclear localization of signal. (D) Total cell lysates from U2OS cells transfected with control vector, HA-FIH 1–349 or HA-FIH delta NES were immunoprecipitated with an anti-exportin 1 antibody. HA-FIH, exportin1 & beta -tubulin levels are indicated. FL, full length; IgGL, IgG light chain. Scale bars: 20 µm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30333145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Phagocyte infiltration contributes to the respiratory burst & consequent EPO signalling activation during inflammation resolution.ZymA (i.p., 1 mg per mouse) was applied to induce peritonitis in male WT mice. (a–c) Mice were treated with either 0.5 mg per mice i.p. of anti-Ly6G antibody or control (isotype control rat IgG2a) 1.5 day before zymA injection, & exudate leucocytes, peritoneum & peritoneal fluid were collected for analysis of ROS (a, n=3), hypoxia, HIF-1 alpha (b, n=3) & EPO (c, n=3) at 6 24 h. (d–f) Mice were treated with either anti-Ly6G antibody (0.5 mg per mice) plus clodronate liposomes (0.2 ml per 10 g) or control (isotype antibody+empty liposome) before zymA injection, & exudate leucocytes, peritoneum & peritoneal fluid were collected for analysis of ROS (d, n=3), hypoxia, HIF-1 alpha (e, n=3) & EPO (f, n=3) at 24 h. Representative data from two independent experiments are shown. For flow cytometry data, black numbers refer to the percentage of positive cells & red numbers refer to the mean fluorescent intensity. Error bars represent the s.e.m. *P<0.05, two-tailed unpaired Student's t-test. Full-size images for b & e are shown in Supplementary Fig. 12. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27397585), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Chromatin Immunoprecipitation: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia reduces inflammatory signaling pathways & NLRP3 expression & induces autophagy in IECs. a HT-29 cells were subjected to normoxia & hypoxia at the indicated times in the absence or presence of 10 µg/ml LPS. Autophagy was measured by variations in the ratio of LC3-II/LC3-I & the total amount of LC3 (LC3-I plus LC3-II) relative to beta -actin. Results are representative of two independent experiments. b, c & d HT-29 cells were subjected to normoxia & hypoxia for the indicated periods in the absence or presence of 10 µg/ml LPS, followed by transcript analysis. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test. Results represent mean + s.e.m. of two independent experiments done in triplicate, *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant. e HT-29 cells were subjected to normoxia or hypoxia in the presence & absence of 10 µg/ml LPS & 20 µM of MG132. Results are representative of two independent experiments. f & g Putative binding sites for HIF-1 alpha & NF-kappa B were found in the p62 f & NLRP3 g promoters using Genomatix software tools. Numbers under the boxes indicate the distance from the transcription start site. HT-29 cells were subjected to normoxia (21% O2) or hypoxia (0.2% O2) for 6 h & 24 h. ChIP analysis was performed using antibodies against HIF-1 alpha & NF-kappa B for immunoprecipitation. PCR was performed using the promoter-specific primers for the p62 f & NLRP3 g promoter binding sites of HIF-1 alpha & NF-kappa B. Aliquots taken prior to immunoprecipitation were used as input control. PCR products were run on 2% agarose gel. The results are representative of three independent experiments. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28740109), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of Hif1a deletion upon growth & invasion. (A) Wild-type (WT) mammary tumor epithelial cells (MTECs) grown to 80% confluence subjected to hypoxic culture for the indicated times up to 24 hours, or cells continued to be cultured under normoxic conditions such that the time t = 0 sample harvested on the same day as the time t = 24 hours hypoxic condition sample. High-salt enriched whole-cell extracts resolved on 3% to 8% Tris-acetate gels & blotted onto polyvinylidene fluoride membrane, which divided horizontally at approximately 60 kDa. The top half of the blot used to detect HIF-1 alpha , & the lower portion used to detect lamin (loading control) to avoid the need to strip & reprobe the blot. (B) Growth curve of WT & knockout (KO) MTECs cultured at normoxia (Nor) or hypoxia (Hyp) in growth medium supplemented w/ 5% fetal bovine serum (FBS) + epidermal growth factor (EGF) (left) or w/ 2% FBS (right). For cells grown in 5% FBS + EGF, a representative graph is shown in which the mean ± SEM of cell number per time point of quadruplicate wells per genotype/oxygen tension is plotted per time point. For cells grown in 2% FBS, the grand mean ± SEM of cell number is presented, which calculated as an average of the mean cell number observed per replicates per time point as observed in three replicate experiments. All data analyzed by two-way analysis of variance (ANOVA, *P < 0.05). (C) The mean fold change (FC) in invasion normalized to invasion index observed for WT cells cultured at normoxia (FC = 1.0). Data represent the mean FC in invasion observed in three independent experiments. All columns compared using one-way ANOVA w/ a Bonferroni posttest. *P < 0.05. Image collected & cropped by CiteAb from the following publication (//breast-cancer-research.biomedcentral.com/articles/10.1186/bcr3087), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Involvement of HIF-1 alpha in alveolar epithelial cell phenotypic changes induced by hypoxia. Primary rat AECs were cultured in normoxia (Nx) (21% O2) or hypoxia (Hx) (1.5% O2) during 6 days in presence or absence of 10 µM YC-1. (A) Immunostaining of ZO-1 (magenta) & TTF1 (cyan) were performed. n = 4 experiments were performed. Isolated primary rat AECs were cultured in normoxia (Nx) (21% O2) or hypoxia (Hx) (1.5% O2) during 6 h in the presence or absence of 100 mM 4-phenylbutyrate (4-PBA) or pre-treated or not with 1 µM BAPTA-AM 90 min before exposition to hypoxia. A representative picture of at least n = 4 independent experiments for each condition has been presented & scale bar represents 50 µm. (B) Western blot of HIF-1 alpha protein levels was performed. Representative blot of n = 5 experiments is shown. Expression levels of HIF-1 alpha were quantified & reported to beta -actin expression for each condition. Primary rat AECs were transfected with plasmid coding for luciferase reporter activity of hypoxia responsive element (HRE: i.e., HIF-luc), & cultured as described. (C) Luciferase activity corresponding to the transcriptional capacity of HIF was reported (n = 4 experiments). Raw data were submitted a Kruskal-Wallis test. * & ** indicate a significant difference as compared with normoxic value with p < 0.05 & p < 0.01 respectively. # & ## indicate a significant difference as compared with value in untreated hypoxic cells with p < 0.05 & p < 0.01, respectively. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30875855), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Pharmacological inhibition of ABCC3 reduces cell proliferation through STAT3 & HIF1 alpha dysregulation & induction of apoptosis. a Representative Western blot images show the effects of pharmacological inhibition of ABCC3 with MCI-715 on the expression of pSTAT3 Y705 & HIF1 alpha in three PDAC cell lines (AsPC1, HPAFII, CFPAC-1). Cells were treated with MCI-715 at the concentration of 10 μM & collected after 24 h (CFPAC-1) or 48 h (AsPC1, HPAFII). The quantitative analysis of n = 3 separate experiments is presented in Additional file 1: Figure S2; b The effects of the treatment of AsPC1, HPAFII & CFPAC-1 cell lines with 10 μM MCI-715 on the Caspase 3/7 activity (72 h post treatment) measured with Caspase 3/7 fluorigenic probe; c Representative Western blotting images & quantitative analysis of cleaved caspase 3 expression following treatment of indicated PDAC cell lines with 10 μM MCI-715. All results are presented as mean ± SEM of 3 independent experiments. The quantitative analysis was performed with the use of ImageJ & Image Lab software, unpaired Student’s t-test was performed for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31378204), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Normoxic pretreatment sensitizes glioma cells to radiation after rapid acute hypoxic (RAH) exposure.(A) The rapid acute hypoxia (RAH) protocol is shown depicting the timing & severity of hypoxic exposure. Cells either remain in a continuous hypoxic environment (–) or are transiently (25 min) exposed to normoxia 25 min prior to radiation (+). Continuously normoxic cells (NOx) were irradiated as a positive control. (B) The results of anchorage-independent colony forming assays are shown for U87, U87-luc, GL261 glioma cells & 0308 GSCs after 5 Gy radiation exposure under varying oxygen conditions. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cell type’s negative (non-irradiated) control & the means & SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (**p<0.01). Also shown are Western blots of nuclear HIF-1 alpha at the time of irradiation for each cell type. Corresponding Western blots of lamin A/C are shown as a loading control. All lanes shown that are non-adjacent to the negative control (NOx) are denoted with a separating black line. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25350400), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Multiple distinct mechanisms for downregulation of AKT activity in TSC1/2-deficient SCs. A-F: Western blots of sciatic nerve lysates from control & TSC2-SCKO/TSC1-SCKO mice at age P28 (A-E) or P14 (F) (3 mice per group), probed with the indicated antibodies. G: Immunofluorescence of longitudinal frozen sciatic nerve sections from control & TSC2-SCKO mice (age P28) showing greatly increased immunoreactivity of PTEN in the mutant (cytoplasmic signals surrounding DAPI+ nuclei). Scale bars: 50 µm H: Schematic summarizing distinct mechanisms underlying the downregulation of AKT activity in SCs from TSC1/2-SCKO nerves. Components highlighted in red are upregulated while blue highlighting depicts downregulation in mutant nerves. Note downregulation of insulin receptor substrate 1 (IRS-1) by constitutively active S6 kinases downstream of mTORC1. This leads to attenuated activation of PI3K/AKT. Lack of mTORC2 stimulation occurs through abolished TSC1/2 which results in reduced AKT phosphorylation in position Ser473 as well as through feedback inhibition by S6 kinases. Lastly, hyperactive mTORC1 promotes Hif1 alpha transcription factor expression which results in increased PTEN transcription, & thus attenuated PI3K/AKT signaling. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29497474), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - DLK1 cleavage is dependent on HIFs & ADAM 17 activity.a, b Representative images & densitometric analysis of western blots showing HIF-1 alpha , HIF-2 alpha , & DLK1 expression & cleavage in U3082MG cells after siRNA targeting of HIF1A & HIF2A in hypoxia. c, d Representative images & densitometric analysis of western blots showing the effects of ADAM inhibition by pre-treatment with 20 µM TAPI-2 on DLK1 cleavage in U3082MG cells grown at 21% or 1% O2 for 48 h. SDHA was used as loading control. e, f Representative images & densitometric analysis of western blots showing the effects of ADAM17 inhibition by pre-treatment with 0.5 μM TMI-1 on DLK1 cleavage in U3082MG cells grown at 21% or 1% O2 for 48 h. SDHA was used as loading control. Statistical analysis: b has 3 independent experiments while d & f have four independent experiments, all data are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by Bonferroni post hoc test. In the whole figure significance is represented as *p < 0.05, **p < 0.01, & ***p < 0.001 vs. respective 21% O2 controls or as indicated by straight lines. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32205867), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia downregulates MHC class I expression via HIF transcription factors.(A-C): siRNA mediated knockdown of HIF-1 alpha reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. MCA205 tumor cells were reverse transfected with scrambled siRNA (NT; red histogram) or with HIF-1 alpha specific siRNA (blue histogram) & cultured as 3D spheroids under 1% (A) or 21% (B) oxygen for 48h. Levels of MHC class I surface expression was determined using flow cytometry. Efficacy of gene knockdown was assessed using Western blot (C). beta -Actin was used as the loading control. Representative data of 3 independent experiments shown. (D-F): Flow cytometry assessment of surface expression of HLA-ABC on paired isogenic renal cell carcinoma cell lines RCC4 (D), UMRC2 (E) & CAKI2 (F). Each pair had the parental cell line that lacked endogenous wild-type VHL (VHL null, transfected with empty vector) & one with vector stably expressing functional VHL (VHL restored). Restoring VHL function & thereby reducing HIF expression, significantly increased HLA-ABC expression on the cells. Representative histograms of 4 independent experiments are shown. Grey filled: unstained control; red: VHL null genotype; blue: VHL restored genotype. (D1-F1): Inactivation of HIF-1 alpha by restoring VHL expression was verified by Western blotting for RCC4 (D1), UMRC2 (E1) & CAKI2 (F1) cells. beta -Actin was used as the loading control. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0187314), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - HIF-1 alpha protein increases in HCT116 cells exposed to rapid intermittent hypoxia. Increased HIF-1 alpha increases HIF-1 transactivation, leading to increased expression of HIF-1 target genes at both the mRNA & protein level. (A) Rapid intermittent hypoxia increases HIF-1 alpha protein after 6 h of exposure. (B) Rapid intermittent hypoxia increases the mRNA expression of genes involved in glycolysis, extracellular matrix remodeling & the HIF pathway after 18 h of exposure. (C) Additional genes with known hypoxia response elements (HRE) binding sites for HIF-1 were measured & all tested genes showed an increase in both chronic hypoxia & intermittent hypoxia after 18 h. All values were normalized to normoxic expression levels, which is equivalent to 0 on the Log2 scale. Results are the mean ± SEM of independent experiments run in duplicate (n ≥ 3). (D) The increase in mRNAs is also reflected in an increase in the protein levels of Glut1, HK2, LDHA & PHD2. * p < 0.05, ** p < 0.01, *** p < 0.001 & **** p < 0.0001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30669593), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hif1 alpha expression in neonatal & adult testis.(A) Section of testis from 5-day old (P5) male new born pups showing Hif1 alpha expression in MVH+ gonocytes within the seminiferous tubules (top) & negative control images without primary antibodies (bottom). Scale bar: 50μm. (B) Western blot analysis of Hif1 alpha expression in P5 testes (left) compared to extract of the adult brain sub-ventricular zone (SVZ) (right). Loading control ( beta -Actin) is shown below. (C) Section of adult (3 month old) testis showing Hif1 alpha expression in spermatogonia. Scale bar: 30μm. (D) Western blot analysis of whole adult testis. HEK293 cells treated with DFX were used as a positive control & intestinal tissue was used as a negative control. Loading control ( beta -Actin) is shown below. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27148974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Roles of P38 mitogen-activated protein kinase (MAPK) in the DFO-mediated up-regulate of HIF-1 alpha protein in APPsw cells. (A) APPsw were exposed to 100 μM Fe, 100 μM DFO, or both combined for 24 h. Whole cell lysates were prepared & subjected to analysis for P38 or GAPDH protein. (B) Western blot analysis revealed that strong phosphorylation of P38 MAPK was detected in the DFO-treated cells, whereas no significant changes were observed in cells treated Fe alone compared with control. There were not statistical changes in the levels of P38 MAPK among the groups. The data represent the mean ± S.E. of three independent experiments. *p < 0.05, **p < 0.01 compared with the control; #p < 0.05, ##p < 0.01 with respect to the Fe treatment group. (C–E) APPsw cells were pretreated without or with SB203580 (10 μM) for 2 h & then treated without or with DFO for additional 22 h in the absence or presence of the inhibitor. Immunoblotting showed that DFO treatment significantly increased the levels of p-P38 MAPK compared with the control group, whereas the increase was significantly ameliorated by SB203580. Furthermore, the DFO-mediated up-regulation of HIF-1 alpha protein was greatly inhibited by SB203580. The data represent the mean ± S.E. (n = 3). **p < 0.01 compared with the control; ##p < 0.01 compared to the values of DFO treatment group (two-way ANOVA with Post hoc Fisher’s PLSD). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26082716), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Expression of hypoxia-inducible factor (HIF) proteins after bilateral renal ischemia or sham ischemia. Immunoblots for HIF-1 alpha (A–C) & HIF-2 alpha (D–F) of tissue extracts from the cortex & outer & inner medulla of the left kidneys of rats 24 h & 5 days following recovery from either sham ischemia (○) or bilateral renal ischemia (●); n = 6 per group. G: typical image of the gel following electrophoresis. H: typical image of the nitrocellulose membrane following transfer. Values are expressed as medians (25th percentile, 75th percentile). Paired comparisons were performed using the Mann-Whitney U-test. Because paired comparisons were made at two time points, P values were conservatively adjusted using the Dunn-Sidak method with k = 2. PTr, PT, & PTr*T are the outcomes of two-way analysis of variance on ranking with the factors treatment (Tr) & time (T). AU, arbitrary unit; I1, 24 h after ischemia; I5, 5 days after ischemia; S1, 24 h after sham ischemia; S5, 5 days after sham ischemia. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30110566), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - DFO regulates the expression of HIF-1 alpha & HIF-1-related protein in the brains of APP/PS1 mice. (A) Western blots showing the expression levels of HIF-1 alpha protein in control & DFO-treated transgenic mice brains. (B) Immunoblot showed that DFO treatment significantly increased HIF-1 alpha protein levels, compared with controls. (C) The expression levels of HIF-1 alpha mRNA were detected by RT-PCR in the brains of APP/PS1 transgenic mice treated with DFO. (D) RT-PCR analysis showed that DFO treatment significantly increased the HIF-1 alpha mRNA levels in the transgenic mouse brain. GAPDH served as the internal control. (E) Immunohistochemically stains showed the distribution of HIF-1 alpha in the cortical & hippocampus sections of APP/PS1 mouse brain (Scale bar = 100 μm). (F) Western blots showing the expression levels of brain-derived neurotrophic factor (BDNF), DMT1, & TFR proteins in APP/PS1 transgenic mice brains 3 months after DFO administration. GAPDH was used as a loading control. (G–J) DFO treatment led to a marked increase in the BDNF, DMT1 + IRE, & TFR protein levels in the brains of transgenic mice compared with the control. There was no significant change in the expression levels of DMT1-IRE protein between the groups, compared with the control. Data represent the mean ± S.E. of 6 independent experiments. **P < 0.01 vs. control group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26082716), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody - BSA Free [NB100-479] - A “white-jade” muscle phenotype in EV-A71-infected mouse strain 129. aLeft panel: We coined a “white-jade” phenotype to describe the pathological changes in the color of the hindlimb muscle in EV-A71-infected mice (indicated by arrow). This phenotype showed characteristic muscle with locally whitened color in appearance & hardened tissue mass. In the saline control, tendons (not muscle) can be seen in white color. Right panel: An anatomical sketch of the hindlimb muscle. GM: gluteal muscle, QF: quadratus femoris muscle, BF: Biceps femoris muscle. b Both VP1 protein (upper) & myogenin protein (lower) were detected by IHC staining in the muscles, indicating EV-A71 infection, muscle injury & regeneration. The magnification is 200X. c Striking expression of HIF1A (hypoxia inducible factor 1-alpha ) protein (brown color) was detected by IHC staining in the white-jaded tissue, but not in the non-white-jaded tissue from the same mouse. d Specific association between HIF1A expression & white-jaded muscle Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31711481), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Kidney-derived Sca-1+ cells produce Epo. (A) Epo protein levels in supernatants obtained from Sca-1+ cells cultured in normoxia or hypoxia (n = 3 biological replicates). (B) HIF-1 alpha & HIF-2 alpha protein levels in Sca-1+ kidney-derived cells after exposure to normoxia (20% O2) or hypoxia (1% O2). Numbers below the blots indicate the fold change of the ratio of HIF-1 alpha /Tubulin & HIF-2 alpha /Tubulin in 1% O2 to the respective 20% O2 control. (C) Epo RNA levels in Sca-1+ cells that were incubated either at 20% O2 of 1% O2 as indicated (n = 4 biological replicates). (D) Mesenchymal stem cell markers in renal Sca-1+ cells on day 21 of culture; reads per kilobase million (RPKM). (E) Volcano plot of 4592 significantly up- & 5376 significantly downregulated mRNAs in kidney-derived Sca-1+ cells on day 21 in culture compared to day 0. Mean values ± SEM are shown. * p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35203399), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Evidence that hypoxia induces nuclear entry of FIH. (A) Protein levels of HIF1 alpha , HIF2 alpha , FIH & PHD2 in U2OS cells during hypoxia (1% O2) treatment at the indicated time points. GAPDH was used as a loading control. (B) Immunofluorescence staining of FIH (green) in U2OS cells under hypoxic conditions (1% O2) at the indicated time points. TO-PRO-3 (blue) was used to stain nuclei. (C) Protein levels of FIH & HIF1 alpha from cytoplasmic or nuclear fractions in U2OS cells in normoxia or hypoxia (1% O2, 3 h). beta -tubulin & PARP were used as loading controls for the cytoplasmic & nuclear fractions, respectively. Figures beneath lanes 2 & 4 indicate relative intensities of nuclear FIH in normoxia & hypoxia. Note that different quantities of cytoplasmic & nuclear extracts were loaded. Scale bars: 20 µm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30333145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Respiratory burst contributes to the EPO signalling activation during inflammation resolution.ZymA (i.p., 1 mg per mouse) was applied to induce peritonitis in male WT mice or CGD mice. (a) Exudate leucocytes & peritoneum were collected at indicated intervals, & protein levels of HIF-1 alpha & HIF-2 alpha were measured by WB (n=3). (b) YC-1, 400083 or 400087 were intraperitoneally given to WT mice 3 h before zymA injection & the protein levels of related molecules were analysed by WB (n=2). (c) Mice were killed 1 h after injection of pimonidazole at indicated intervals & the protein adducts of reductively-activated pimonidazole were detected by WB with Hypoxiprobe-1-Mab-1 (HPI) (n=2). (d) Flow cytometry for ROS at indicated intervals (n=3). (e) Flow cytometry for cellular sources of ROS at indicated intervals (n=3). Representative data from at least two independent experiments are shown. For flow cytometry data from d, black numbers refer to the percentage of positive cells & red numbers refer to the mean fluorescent intensity. Error bars represent the s.e.m. *P<0.05, two-tailed unpaired Student's t-test. Full-size images for a–c are shown in Supplementary Figs 8–10, respectively. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27397585), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Effect of IKK2 inhibition on HUVEC, microvessel outgrowth from aortic rings, & VEGF-A expression. a Dose-dependent (10, 5 & 2.5 ng/ml) inhibitory effect of IMD0354 on HUVEC migration relative to control (DMSO 1 µl/ml). Living HUVEC were visualized with Calcein-AM (green). b Quantification of HUVEC migration distance (n = 8). One-way ANOVA test with Tukey multiple comparison was used to determine statistical significance. c HUVEC has grown on Geltrex to evaluate tube formation in the presence of IMD0354 compared to drug-free vehicle (DMSO). Vital HUVEC are stained with calcein-AM (green), & dead HUVEC are displayed in red (propidium iodide). Quantitative analysis of a number of junctions (d), & tubules (e) formed by HUVEC, & total tubule length (f) (n = 8). g Quantitative analysis of cell death induced by IMD0354 treatment (n = 8). h Effect of IMD0354 on cell proliferation in the aortic ring assay. Student t test was used to determine statistical significance. i Western blot analysis of VEGF-A expression in HUVEC treated with IMD0354 (10, 5, 2.5 ng/ml), with beta -actin as a loading control. j Western blot analysis of HIF-1 alpha expression in HUVEC treated with IMD0354 (10, 5, 2.5 ng/ml), with beta -actin as a loading control. k Immunofluorescent detection of VEGF-A (green) in HUVEC treated with IMD0354 (10, 5, 2.5 ng/ml). Cell nuclei visualized with DAPI staining (blue). n.s. p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 Image collected & cropped by CiteAb from the following publication (//link.springer.com/10.1007/s10456-018-9594-9), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - The duration of enhanced radiosensitivity after normoxic pretreatment of cells undergoing Rapid Acute Hypoxia.(A) The RAH protocol is shown with variable delays to radiation after normoxic pretreatment. In this protocol all cells are transiently (25 min) exposed to normoxia for 25 min (+) & then returned to severe hypoxia (1% O2) for 1, 3, or 6 hours prior to radiation. Continuously normoxic cells (NOx) were irradiated as a positive control. (B) Results from anchorage-independent colony forming assays indicate that the decay of enhanced radiosensitivity for cells in the RAH protocol is generally more rapid than that observed for cells in the GCH protocol. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cell type’s negative (non-irradiated) control & the means & SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p<0.05, **p<0.01). Also shown are Western blots of nuclear HIF-1 alpha at the time of irradiation for each cell line. Corresponding Western blots of lamin A/C are shown as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25350400), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - HIF-1 alpha overexpression rescues oxygen-induced radioresistance in RAH-treated cells, but not GCH-treated cells.Results are shown for the anchorage-independent colony forming assays for U87 cells transfected with either an empty vector or HIF-1 alpha expression vector & then exposed to GCH or RAH protocols without (–) or with (+) reoxygenation. Continuously normoxic cells (NOx) were irradiated as a positive control. To allow for ease of comparisons among conditions, raw values are presented as a percentage of that cell type’s negative (non-irradiated) control & the means & SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p<0.05, **p<0.01). Western blotting analysis of nuclear HIF-1 alpha at the time of irradiation is shown for each cell type below clonogenic results. Corresponding Western blots of lamin A/C are shown as a loading control & blots for hemagglutinin (HA) are shown below HIF-1 alpha overexpression vector results to demonstrate transfection efficacy. All lanes shown that are non-adjacent to the negative control (NOx) are denoted with a separating black line. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25350400), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - ONECUT2 modulates HIF1 alpha binding to chromatin in NE-like PC3 cells. a Western blot of HIF1 alpha & ONECUT2 in PC3 cells under normoxic & hypoxic conditions. Two different siRNAs targeting ONECUT2 were mixed together for knockdown experiments. b Left panel: heatmaps show HIF1 alpha ChIP-Seq signal in PC3 cells under hypoxic conditions with & without knockdown of ONECUT2; right panel: pileup of HIF1 alpha ChIP-Seq signals centered at HIF1 alpha ChIP-Seq peaks center. c Expression of ANGPTL4 & ADM, two hypoxia-regulated genes, with & without knockdown of ONECUT2 in PC3 cells. d Schematic illustration of the analysis identifying SMAD3 as an ONECUT2 regulated HIF1 alpha co-factor. Genes identified in motifs enriched in HIF1 alpha binding sites & ONECUT2 target genes were further filtered by HIF1 alpha interacting protein list from BioGRID. ONECUT2 target genes were defined as differentially expressed in ONECUT2 knockdown & control samples & with ONECUT2 binding sites nearby in PC3 cells under hypoxic conditions. e SMAD3 expression in response to ONECUT2 silencing in PC3 cells. f SMAD3 & HIF1 alpha binding sites with & without silencing of SMAD3. g The overlap of SMAD3 ChIP-Seq, HIF1 alpha ChIP-Seq & SMAD3-HIF1 alpha ChIP-re-ChIP-Seq peaks. h SMAD3 ChIP-Seq, HIF1 alpha ChIP-Seq & SMAD3-HIF1 alpha ChIP-re-ChIP-Seq signal near the promoter regions of hypoxia-induced genes ANGPTL4 & ADM. Error bars indicate s.d. from at least two technical replicates. P-value is calculated by one-way ANOVA. **: P < 0.01. Source data are provided as a Source Data file Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30655535), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - The duration of enhanced radiosensitivity after normoxic pretreatment differs among cell lines undergoing Graded Chronic Hypoxia.(A) The GCH protocol is shown with variable delays to radiation after normoxic pretreatment. After GCH, all cells are transiently (25 min) exposed to normoxia for 25 min (+) & are then returned to severe hypoxia (1% O2) for 1, 3, or 6 hours prior to radiation. Continuously normoxic cells (NOx) were irradiated as a positive control. (B) Results from anchorage-independent colony forming assays indicate that the decay of enhanced radiosensitivity differs among cell lines. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cell type’s negative (non-irradiated) control & the means & SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p<0.05, **p<0.01). Also shown are Western blots of nuclear HIF-1 alpha at the time of irradiation for each cell line. Corresponding Western blots of lamin A/C are shown as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25350400), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - G9a inhibition reduces cell motility in breast cancer cell lines. G9a inhibition represses breast cancer cell motility in vitro. (A) Microarray analysis of differentially expressed genes comparing hypoxia-responsive genes from MCF7 cells expressing shNS & shG9a identifies a subset of 212 genes downregulated in hypoxia in a G9a-dependent manner. (B) Ingenuity Pathway Analysis results of the 212 G9a-dependent genes. (C) Evaluation of the migratory distance covered by MDA-MB-231 treated with UNC0642 (5 μM) or (D) transfected with either vector or shG9a. Results were evaluated through real-time imaging using the HoloMonitor M4, taking pictures every 10 minutes for 48 hours, in three independent experiments. An average of 20 cells per condition is shown for the representative circular displacement images. (E) Western blot analysis of G9a, H3K9me1 & H3K9me2 in MDA‑MB-231 transfected with vector or shG9a & incubated in hypoxia for 24 hours. HIF1 alpha was included as positive control for hypoxic conditions. H3 & Lamin A/C were used as loading control (F) Scratch wound assay for MDA-MB-231 breast cancer cells treated with 5 μM UNC0642, under both normoxic (21% O2) & hypoxic (1% O2) conditions. Results were evaluated by real-time imaging performed by the IncuCyte Zoom every 24 hours & wound closure was quantified using ImageJ. Scale bar represents 500 μm. (G) Scratch wound assay of MCF7 breast cancer cells following G9a KD & G9a reconstitution. Scale bar represents 500 μm. (H) Western blot analysis of MCF7 cells transfected with shG9a & reconstituted with WT G9a. Data are represented as mean ± SEM of three independent experiments (unpaired, non-parametric Student's t-test, **p<0.005, ****p<0.0001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32292512), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Deletion of Hif1 alpha decreases primary tumor growth. (A) Wild-type (WT) or knockout (KO) cells (n = 50,000) were transplanted into FVB/Nj mice. All tumors were harvested at day 56 to evaluate tumor weight, volume & burden (percentage tumor weight/total body weight, BW) (n = 10 recipients/genotype, P < 0.05, unpaired Student's t-test). (B) The growth rate of WT & KO tumors following transplant of 50,000 cells. Best-fit curves were established based on a polynomial fit algorithm using GraphPad Prism 4.0 software. Data in (A) & (B) are representative of seven independent experiments (> 60 recipients/genotype). (C) When 500 WT & KO cells were implanted into the mammary fat pad, the median time until 50% of recipients developed tumors > 500 mm3 was 64 days for WT mice & 127 days for KO mice (n = 14 recipients/genotype, P < 0.001, logrank test). (D) Western blot for HIF-1 alpha in three independent tumors (500 to 750 mm3) per genotype. WT & KO cells that were cultured for 6 hours under hypoxic conditions served as positive & negative controls, respectively. CRM, cross-reactive material. MTEC, mammary tumor epithelial cell. (E) Mean fold change ± SEM in expression of HIF-1 targets in KO tumors as determined by qRT-PCR (n = 5 tumors/genotype). Decreased gene expression in KO tumors is presented as a negative fold-change relative to WT tumors. (F) An increase in Ki67+ cells in KO tumors is balanced by an increase in caspase 3-positive cells (n = 5 tumors/genotype, *P < 0.05, Student's t-test). Representative immunostaining images are shown in Additional file 2 Figure S4. Image collected & cropped by CiteAb from the following publication (//breast-cancer-research.biomedcentral.com/articles/10.1186/bcr3087), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hif1 alpha expression in neonatal & adult testis.(A) Section of testis from 5-day old (P5) male new born pups showing Hif1 alpha expression in MVH+ gonocytes within the seminiferous tubules (top) & negative control images without primary antibodies (bottom). Scale bar: 50μm. (B) Western blot analysis of Hif1 alpha expression in P5 testes (left) compared to extract of the adult brain sub-ventricular zone (SVZ) (right). Loading control ( beta -Actin) is shown below. (C) Section of adult (3 month old) testis showing Hif1 alpha expression in spermatogonia. Scale bar: 30μm. (D) Western blot analysis of whole adult testis. HEK293 cells treated with DFX were used as a positive control & intestinal tissue was used as a negative control. Loading control ( beta -Actin) is shown below. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27148974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Hypoxia reduces inflammatory signaling pathways & NLRP3 expression & induces autophagy in IECs. a HT-29 cells were subjected to normoxia & hypoxia at the indicated times in the absence or presence of 10 µg/ml LPS. Autophagy was measured by variations in the ratio of LC3-II/LC3-I & the total amount of LC3 (LC3-I plus LC3-II) relative to beta -actin. Results are representative of two independent experiments. b, c & d HT-29 cells were subjected to normoxia & hypoxia for the indicated periods in the absence or presence of 10 µg/ml LPS, followed by transcript analysis. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test. Results represent mean + s.e.m. of two independent experiments done in triplicate, *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant. e HT-29 cells were subjected to normoxia or hypoxia in the presence & absence of 10 µg/ml LPS & 20 µM of MG132. Results are representative of two independent experiments. f & g Putative binding sites for HIF-1 alpha & NF-kappa B were found in the p62 f & NLRP3 g promoters using Genomatix software tools. Numbers under the boxes indicate the distance from the transcription start site. HT-29 cells were subjected to normoxia (21% O2) or hypoxia (0.2% O2) for 6 h & 24 h. ChIP analysis was performed using antibodies against HIF-1 alpha & NF-kappa B for immunoprecipitation. PCR was performed using the promoter-specific primers for the p62 f & NLRP3 g promoter binding sites of HIF-1 alpha & NF-kappa B. Aliquots taken prior to immunoprecipitation were used as input control. PCR products were run on 2% agarose gel. The results are representative of three independent experiments. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28740109), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - Transient hyperoxia does not sensitize normal human astrocytes to radiation.Results are shown for the colony forming assays for all previously assayed cell lines & a normal human astrocyte cell line (Astro). Cells were continuously maintained under normoxic conditions (NOx) or exposed to 25 min of hyperoxia (50% O2) & then returned to normoxic conditions for 25 min (HyperOx) before being treated with a 5 Gy dose of radiation. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cells type’s negative (non-irradiated) control & the means & SEMs are plotted. Each result represents three independent samples, plated in triplicate (*p<0.05, Student’s t-test). Also shown are Western blots of nuclear HIF-1 alpha at the time of irradiation for each cell line. Corresponding Western blots of lamin A/C are shown as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25350400), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: HIF-1 alpha Antibody - BSA Free [NB100-479] - YC-1 attenuates pro-inflammatory differentiation of macrophages. (a,b) qRT-PCR analyses, revealing mRNA expression of the pro-inflammatory M1-differentiation markers NOS2 (a) & IL-6 (b) in murine J774A.1 macrophages under normoxia (left bars) & hypoxia (right bars). LPS exposure was carried out to induce M1-polarization in presence of YC-1 or vehicle control (DMSO; **P ≤ 0.01, n = 9). (c,d) Western blots of nuclear extracts, revealing protein levels of HIF-1 alpha (c) & HIF-2 alpha (d) in J774.A1 macrophages treated with vehicle (DMSO) or YC-1 under normoxic (left) or hypoxic (right) culture conditions. Simultaneous LPS-treatment was performed to induce pro-inflammatory activation. Note LPS-induced stabilization of HIF-1 alpha - (c), but not HIF-2 alpha -levels (d), & reversal of LPS-induced HIF-1 alpha -stabilization by YC-1 (c). Histone H3 was used as a loading control. (e,f) qRT-PCR analyses, revealing mRNA expression of the pro-inflammatory M1-differentiation markers NOS2 (e) & IL-6 (f) in BMDMs under normoxia (left bars) & hypoxia (right bars). LPS exposure was carried out to induce M1-polarization in presence of YC-1 or vehicle control (DMSO; ***P ≤ 0.001, **P ≤ 0.01, n = 6). Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-13638-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Ca, Fi, Gt, Ha, Pm, Rb, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Ca, Fi, Gt, Ha, Pm, Rb, Ze

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB100-479AF350
Alexa Fluor 405	NB100-479AF405
Alexa Fluor 488	NB100-479AF488
Alexa Fluor 532	NB100-479AF532
Alexa Fluor 594	NB100-479AF594
Alexa Fluor 647	NB100-479AF647
Alexa Fluor 700	NB100-479AF700
Alexa Fluor 750	NB100-479AF750
Alexa Fluor Plus 405	NB100-479AFP405
Alexa Fluor Plus 488	NB100-479AFP488
Alexa Fluor Plus 555	NB100-479AFP555
Alexa Fluor Plus 594	NB100-479AFP594
Alexa Fluor Plus 647	NB100-479AFP647
Alexa Fluor Plus 680	NB100-479AFP680
Allophycocyanin	NB100-479APC
Allophycocyanin/Cy7	NB100-479APCCY7
Biotin	NB100-479B
CoraFluor 1	NB100-479CL1
DyLight 350	NB100-479UV
DyLight 405	NB100-479V
DyLight 488	NB100-479G
DyLight 550	NB100-479R
DyLight 594	NB100-479DL594
DyLight 650	NB100-479C
DyLight 680	NB100-479FR
DyLight 755	NB100-479IR
FITC	NB100-479F
HRP	NB100-479H
Janelia Fluor 525	NB100-479JF525
Janelia Fluor 549	NB100-479JF549
Janelia Fluor 646	NB100-479JF646
Janelia Fluor 669	NB100-479JF669
PE	NB100-479PE
PE/Atto594	NB100-479PEATT594
PE/Cy5.5	NB100-479PECY55
PE/Cy7	NB100-479PECY7
PerCP	NB100-479PCP
mFluor Violet 450 SE	NB100-479MFV450
mFluor Violet 500 SE	NB100-479MFV500
mFluor Violet 610 SE	NB100-479MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB100-479SS

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AF1924

SOX17 Antibody [Unconjugated]

(3 Reviews)

(557 Publications)

100 μg

AF1924

HAF109). A specific band was detected for SOX17 at approximately 55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_lightbox">

<b>Biological Strategies Validation. </b>Western blot shows lysates of BG01V human embryonic stem cells untreated (-) or endoderm differentiated (+). PVDF membrane was probed with 1 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (<a class=

HAF109). A specific band was detected for SOX17 at approximately 55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." alt="Western blot shows lysates of BG01V human embryonic stem cells untreated (-) or endoderm differentiated (+). PVDF membrane was probed with 1 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF109). A specific band was detected for SOX17 at approximately 55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." class="big_thumb" />

AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (BAF109). Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (MAG999) and DNA was purified using chelating resin solution. Thep21promoter was detected by standard PCR." class="big_lightbox">

<b>Biological Strategies Validation. </b>Endoderm-differentiated D3 mouse embryonic stem cell line was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. SOX17/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) or control antibody (<a class=

AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (BAF109). Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (MAG999) and DNA was purified using chelating resin solution. Thep21promoter was detected by standard PCR." class="big_thumb" />

NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_lightbox">

SOX17 was detected in immersion fixed B16 mouse melanoma cell line using 10 µg/mL Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; <a class=

NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_thumb" />

NL001) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_lightbox">

SOX17 was detected in immersion fixed endoderm differentiated BG01V human embryonic stem cells using 10 µg/mL Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red, upper panel; <a class=

NL001) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips." class="big_thumb" />

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_lightbox">

<b>Biological Strategies Validation. </b>Simple Western lane view shows lysates of BG01V human embryonic stem cells untreated (-) or endoderm differentiated (+), loaded at 0.2 mg/mL. A specific band was detected for SOX17 at approximately 59 kDa (as indicated) using 10 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # <a class=

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_thumb" />

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_lightbox">

<b>Biological Strategies Validation. </b>Simple Western lane view shows lysates of iBJ6 human induced pluripotent stem cell line untreated (-) or endoderm differentiated (+), loaded at 0.2 mg/mL. A specific band was detected for SOX17 at approximately 58 kDa (as indicated) using 10 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # <a class=

HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system." class="big_thumb" />

HAF017). A specific band was detected for SOX17 at approximately 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1." class="big_lightbox">

Western blot shows lysates of SK‑OV‑3 human ovarian adenocarcinoma cell line, OVCAR‑3 human ovarian carcinoma cell line. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # <a class=

HAF017). A specific band was detected for SOX17 at approximately 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1." class="big_thumb" />

af1924_human-sox17-affinity-purified-polyclonal-ab-21202416502.jpg

af1924_human-sox17-affinity-purified-polyclonal-ab-21202416508.jpg

af1924_human-sox17-affinity-purified-polyclonal-ab-21202416501.jpg

af1924_human-sox17-affinity-purified-polyclonal-ab-21202416500.jpg

af1924_human-sox17-affinity-purified-polyclonal-ab-21202416504.jpg

<b>Biological Strategies Validation. </b>af1924_human-sox17-affinity-purified-polyclonal-ab-212024165016.jpg

af1924_human-sox17-affinity-purified-polyclonal-ab-21202416506.jpg

<b>Biological Strategies Validation. </b>af1924_human-sox17-affinity-purified-polyclonal-ab-21202416503.jpg

af1924_human-sox17-affinity-purified-polyclonal-ab-21202416505.jpg

Characterization of hybrid cells.(A) Both ES-pNSC and pES-NSC hybrid cells are positive for alkaline phosphatase staining (100 ×). (B) RT-PCR analysis of Oct4, Nanog, Sox2, and Nestin expression in fusion partner and reprogrammed hybrid cells. Pluripotency markers, Oct4 and Nanog, which were not expressed in NSCs and pNSCs were expressed in GFP+ fusion hybrid cells. On the other hand, Nestin, which was expressed in NSCs and pNSCs was silenced after forming GFP+ fusion hybrid cells. (C) Immunocytochemistry analysis of Oct4 and Nanog in ES-pNSC and pES-NSC hybrid cells (100 ×). (D)In vitro differentiation of ES-pNSC and pES-NSC hybrid cells into ectoderm (Tuj1), mesoderm (SMA), and endoderm (Sox17) lineages (200 ×). (E) In vivo differentiation potential of ES-pNSC and pES-NSC hybrid cells through teratoma assay. These hybrid cells were contributed to secretory epithelium (ectoderm), cartilage (mesoderm) and gut epithelium (endoderm), which were stained with PAS, Asian blue, and hematoxylin eosin, respectively. Each tissue was indicated by arrow head. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0156491), licensed under a CC-BY license. Not internally tested by R&D Systems.

In vitro and in-TiPSCs.(A): Immunofluorescence staining for Sox17 (endodermal marker), alpha SMA (mesodermal marker), and Nestin (ectodermal marker) in each TiPSCs1-derived differentiated cell in vitro. (B): Gross morphology of representative teratomas derived from TiPSCs1 in vivo (hematoxylin and eosin staining). Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0097397), licensed under a CC-BY license. Not internally tested by R&D Systems.

Activation of Sox17 at onset of EC regeneration and Sox17-mediated Cyclin E1 expression. a qPCR analysis of gene expression in sorted CD31+ cells from mTmG-Scl mice before and after injury (12 mg/kg i.p.). Sox17, Vegfr2, and Ccne1 increased significantly at day 2 post-LPS compared to baseline. n = 3. Color scale: the fold change increases from red to white to green color. b Western blot analysis in fresh isolated ECs from wild-type mice and quantification c showed a 5-fold increase in Sox17 protein expression within 1 day following injury compared to baseline and followed by recovery within 3 days post-LPS. n = 3. d, e Western blot analysis of cultured HLMVECs in which Sox17 was overexpressed showed 2.5x fold increase in Cyclin E1 protein expression relative to control cells. n = 3. OE, overexpression. f Representation of the CCNE1 promoter region with Sox17 binding sites (circled numbers) and their sequences. g HLMVECs were retrovirally transduced with Sox17 or control plasmid for 3 days, and Ch-IP assay followed by qPCR was performed to amplify Sox17 binding sites in the CCNE1 promoter. n = 3. h 293T cells were transfected with a Sox17 overexpression plasmid containing CCNE1 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (c) and two-way ANOVA with Bonferroni post-tests for (e, g, h) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

Generation and characterization of systemic lupus erythematosus (SLE)‐specific human‐induced pluripotent stem cells (hiPSCs). (A) Dermal fibroblasts derived from patient with SLE were reprogrammed into iPSCs using Sendai virus vectors and three clones (#1, #2 and #3) were characterized. RT‐PCR confirms the loss of transgenes in hiPSCs‐SLE (lanes 1, 2 and 3), presence (lane 4) in infected fibroblasts (ipF‐SLE) and absence of Sendai viral transgenes in parental fibroblasts (pF‐SLE) (lane 5). Full‐length gels are presented in File S2. (B) The hiPSCs‐SLE colonies expressed alkaline phosphatase. Scale bar, 500 μm. (C) RT‐qPCR analysis of pluripotency genes OCT4, NANOG, SOX2 and REX1 was performed in fibroblasts and in hiPSCs derived from patient with SLE. All expression values are normalized to GAPDH and relative donor fibroblasts. Data are mean ± SEM and all statistical analysis was made between hiPSCs‐SLE clones and relative fibroblasts by Student's t test showing P‐values ≤ .05 in each comparison. (D) PluriTest assays combines novelty score (blue) on x‐axis and pluripotency score (red) on y‐axis. hiPSCs‐SLE localize in the red cloud suggesting the empirical distribution of pluripotent cells compared to non‐pluripotent blue cloud. (E) Representative images of M‐FISH staining show normal karyotypes of hiPSCs‐SLE clones. (F) Immunofluorescence analysis of pluripotent stem cell markers Nanog (green), Oct4 (red) and co‐staining with DAPI (blue) in hiPSCs‐SLE. Scale bar, 50 μm. (G) Representative images of floating and adherent EBs derived from hiPSCs‐SLE at differentiation day 8 and 18, respectively. Scale bar, 500 μm. (H) RT‐qPCR results confirm the capability of hiPSCs‐SLE to differentiate into all three germ layers. The expression levels of GATA4, HAND1 and PAX6 in EBs are relative to undifferentiated hiPSCs. All expression values are normalized to GAPDH and relative hiPSCs. Data are mean ± SEM and all statistical analysis was made between EBs‐SLE and relative hiPSCs‐SL

soHIF-1 alpha signaling induces Sox17 expression. a MPO activity of flushed lung sample from mice challenged with LPS (12 mg/kg i.p.) for 6 and 24 h. n = 3. b Western blot analysis of wild-type mice lung before and after LPS-induced injury (12 mg/kg i.p.) and its quantification c showed that HIF-1 alpha protein expression increased within 6 h post-LPS and remained increased until day 2. n = 3. d Western blot analysis in freshly isolated ECs from Hif1afl/fl mice and quantification e showed significantly increased Sox17 protein expression after injury when compared to baseline levels. n = 3. f Western blot analysis in freshly isolated ECs from Hif1aEC−/− mice and quantification g showed no significant difference in Sox17 protein expression before and after injury. n = 3. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (b, c, e, g). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

PGCLCs derived from hc-iPSCs.Immunofluorescent staining of (A) BLIMP1 (green color) and (B) OCT4 (green color) in 4-day-old EBs cultured with PGC induction medium on hc-iPSC-1, -2, -5 and -6 (third to fifth column), PBMC derived iPSC0102 and iPSC0207 (right two column). H9 hESCs (left column) adapted in 4i medium was also detected for BLIMP1 and OCT4 antibodies. Noted that there was no BLIMP1 signals detected in undifferentiated H9 in 4i condition. All samples were doubly stained with SOX17 antibody (red color) sequentially. DAPI was used for counterstain (blue). Scale bar = 50μm. (C) Quantification of immunofluoresent staining of PGCLCs. BLIMP1/SOX17 and OCT4/SOX17 double positive cells were counted for PGCLCs and normalized with the number of DAPI. Percentage of PGCLCs per EB of all lines was presented and significance was compared with control hESC H9. ** indicates P<0.005. *** indicates P<0.001. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0165715), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>Activin-A levels during Day 0–1 modulate CM vs. DE differentiation.Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. Panel A, a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); e is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. Panel A f-i shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. Panel B depicts the effect of Activin-A levels during Day 0–1 on cardiomyocyte differentiation at Day 14, determined by flow cytometry using anti-cTnT. Cultures treated with 10 ng/ml Activin-A began to rhythmically contract at Day 6. Cells treated with 50 or 100 ng/ml Activin-A did not beat at any time. Bars indicate the average values combined from multiple experiments. Vertical lines = ±SEM. P-values were calculated by Student’s t-test. The p-value over the bar denoting 10 ng/ml Activin-A is relative to cells treated with CHIR only (0 ng/ml Activin-A), whereas the p-values over the bars denoting 50 and 100 ng/ml Activin-A are relative to cells treated with 10 ng/ml Activin-A. The size bar in Aa, which pertains to panels a-i, = 200 μm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0118670), licensed under a CC-BY license. Not internally tested by R&D Systems.

Features of ESC-INTPSCs intermediate between ESCs and EpiSCs.(A) Comparison of gene expression in ESCs, EpiSCs, and ESC-INTPSCs. (B) The number of AP-positive colonies formed in the LIF/2i condition four days after 1000 cells were seeded per dish. (C) Proliferation of ESCs, EpiSCs, and ESC-INTPSCs with or without JAKi. (D) Expression of ESRRB and FOXA2. White arrows indicate ESRRB- and FOXA2-double–positive cells. Scale bar, 100 µm. (E) Percentage of ESRRB-positive or ESRRB- and FOXA2-double–positive cells in ESCs, ESC-INTPSCs, and EpiSCs. (F) Expression of OCT3/4 and SOX17. Scale bar, 100 µm. (G) The number of Oct3/4-GFP–positive or –negative colonies derived from single cells at day 4 and 8. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0095329), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>EC specific deletion of Sox17 (Sox17EC−/−) in mice prevents endothelial regeneration. a Schematic diagram of Sox17fl/fl mice crossed with Scl-CreERT2 to delete Sox17 in ECs. After tamoxifen feeding for five days and rest for another four weeks, mice are challenged with LPS (sub-lethal 8 mg/kg, i.p.) for analysis. b Western blot analysis of Sox17 protein expression in isolated ECs obtained from flushed lungs of Sox17EC−/− and control mice. n = 4. c Quantification of b shows 80% deletion of Sox17 in ECs of Sox17EC−/− mice compared to control mice. n = 4. d Time course of lung transvascular permeability following LPS challenge in Sox17EC−/− and Sox17fl/fl mice. n = 4. While control mouse lungs showed increased endothelial permeability at day 1 post-LPS and then recovered to baseline by day 5, Sox17EC−/− mice showed prolonged endothelial barrier leakiness post-LPS. e Time course of changes in lung transvascular permeability following LPS challenge was also carried out in Sox17fl/fl mice crossed with Cdh5-CreERT2 mice. n = 4. Similar as in d, these Sox17EC−/− mice also showed persistent leakiness post-LPS while control mice fully recovered. f Survival curve of LPS challenge in Sox17EC−/− and control mice. n = 8 per group. At this sub-lethal dose, all control mice survived whereas half of Sox17EC−/− mice died on day 2 post-LPS with increased mortality on day 3. By day 5, the death rate for control mice is 0 while for Sox17EC−/− mice is 60%. g Flow cytometry analysis of CD31+CD45− ECs among whole lung population in mice following injury. n = 4. In contrast to control mice in which CD31+CD45− EC population gradually recovered with day 3 post-LPS after initial loss of ECs, Sox17EC−/− mice showed significantly delayed restoration of ECs post-LPS period. h Quantification of BrdU+ nuclei in each field of 425 μm2 area in flushed lung cryo-sections from mice following injury. n = 4 mice per group and 6 replicates per sample. Slides are co-stained with CD31-AF488, BrdU-APC, and DA

<b>Biological Strategies Validation. </b>p38-Mapk14/11 inhibition during blastocyst maturation blocks PrE differentiation/maturation. (a) Experimental schema of p38-Mapk14/11 inhibition (+SB220025), plus vehicle control (+DMSO), and the details of antibodies used to analyse ICM cell lineage marker protein expression by immunofluorescence (IF) in late blastocysts (E4.5); Nanog and Gata4 (+DMSO n = 27, +SB220025 n = 33)—green, Nanog and Sox17 (+DMSO n = 18, +SB220025 n = 20)—red, and Nanog and Gata6 (+DMSO n = 24, +SB220025 n = 27)—blue. (b) Representative single confocal z-plane micrographs of vehicle control-treated (+DMSO) or p38-Mapk14/11 inhibited (+SB220025) late-blastocyst stage/equivalent embryos, immunofluorescently stained for indicated ICM cell lineage markers (Nanog in green and Gata4, Sox17 and Gata6 in red, plus DAPI DNA stain in blue). Examples of cells classified as TE, PrE and EPI are marked with an asterisk, arrowhead and arrow, respectively. Scale bar, 15 µm. (c) Pie charts of the relative cell lineage contribution in vehicle control (+DMSO) and p38-Mapk14/11 inhibited (+SB220025) blastocysts as judged by IF to detect the stated ICM lineage marker proteins. Blue, trophectoderm (TE); yellow, epiblast (EPI—ICM exhibiting exclusive Nanog expression); green, primitive endoderm (PrE—ICM exhibiting exclusive Gata4/6 or Sox17 expression, as appropriate); orange, uncommitted ICM cells (exhibiting co-expression of both Nanog and Gata4/6 or Sox17, as appropriate); and grey, ICM cells negative for either assayed marker. (d) Bar charts show average number of cells allocated to each specified ICM lineage, as judged by the indicated IF staining regime employed. Error bars represent s.e.m. and asterisks denote statistical significant differences in cell number between the vehicle control (+DMSO, black bars) and p38-Mapk14/11 inhibited (+SB220025, grey bars) embryo groups, according to two-tailed Student's t-test, with *p < 0.05 and **p < 0.005 confidence intervals. Yellow asterisk denotes increase in cel

<b>Biological Strategies Validation. </b>Differentiation of hESCs into definitive endoderm cells by Activin A with CHIR99021.a The relative primitive streak (BRACHYRUY, MESP1, and MIXL1) gene expression of the day 1 differentiated cells by adding Activin A (100 ng/mL) with CHIR99021 (2–5 µM) or Wnt3a (25–100 ng/mL) treatments were determined by real-time quantitative PCR (qPCR). None, no WNT signaling pathway activators were used on day 1 for PS differentiation (Activin A only). b, c After 1 day, the medium was changed to Activin A (100 ng/mL) and 1 × CTS-B27 to induce DE differentiation. The definitive endoderm (FOXA2 and SOX17) and mesoderm (MESP2 and HAND1) relative gene expression levels were determined by qPCR. d Immunofluorescence analysis of the expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells on day 3. e, f The expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells was determined by flow cytometry on day 3. Isotype control antibodies were used as controls. At a specific gene expression, datum points lacking common letters differ, p < 0.05. Data are represented as the mean ± SD. Scale bar, 100 µm Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31601782), licensed under a CC-BY license. Not internally tested by R&D Systems.

<b>Biological Strategies Validation. </b>HIF-1 alpha activates transcription of Sox17. a Western blot analysis in control HLMVECs and HLMVECs for which CRISPR/Cas9 was used to delete HIFs. ECs were treated with the HIF prolyl hydroxylase inhibitor DMOG to induce HIF expression. DMOG (1 mM) increased HIF-1 alpha and HIF-2 alpha protein expression in control ECs but not in ECs lacking HIF-1 alpha or HIF-2 alpha . Induction of HIF expression was coupled to Sox17 upregulation. n = 3. b Quantification of a showed that protein expression of HIF-1 alpha and HIF-2 alpha in ECs was significantly increased by DMOG treatment. Sox17 showed a 2.5-fold increase in Sox17 expression control DMSO treated ECs. The increase in Sox17 was significantly reduced in HIF-1 alpha -deleted ECs but preserved in HIF-2 alpha -deleted ECs, indicating the importance of HIF-1 alpha in mediating Sox17 expression. n = 3. c Representation of the SOX17 promoter region with 3 HREs indicated by circled numbers, and their respective sequences are displayed. d HLMVECs were exposed to normoxia or 1% O2 (hypoxia) for 8 h. Ch-IP assay followed by qPCR was performed to amplify the HRE regions in the SOX17 promoter. Studies were performed in ECs exposed to either normoxia or hypoxia. n = 3. e 293T cells were transfected with a HIF-1 alpha overexpression plasmid containing SOX17 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. Results show that hypoxia activation of SOX17 HRE3 was required for Sox17 expression. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for (b, d, e) Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

Concordant expression of developmental programs across organoids from four human iPSC lines. a Heatmap of expression patterns for major nephrogenesis markers across organoid differentiation time points (iPSC D0, D7, D15, and D29, averaged across four cell lines, ML protocol) and human adult kidney. Expression values were row-normalized to obtain z-scores; red color indicates positive z-scores. b Canonical (NPHS2) and data-derived (CLDN5) podocyte marker genes superimposed in tSNE plots from D15 organoids (N2 line, ML protocol). c IF staining of D15 kidney organoid (N2 line, ML protocol) for CLDN5 as a marker of early podocyte differentiation derived from the single-cell data. Additional canonical podocyte markers (NPHS1, WT1) and DAPI staining as shown. d IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31784515), licensed under a CC-BY license. Not internally tested by R&D Systems.

Localisation of Sox17 staining in proliferative and secretory phase endometrial tissue and regulation by hormones. Within the proliferative (A) and secretory (B) endometrium, Sox17 localized to the glandular (open arrowheads) and luminal (closed arrowheads) epithelium, with staining appearing in an irregular, patchy, pattern. Smaller inset boxes show relevant IgG negative controls. Imaged at 20x magnification, scale bar = 50 μm. Treatment of endometrial luminal epithelial (ECC-1) cells with 10−8 M estrogen (estradiol)/10−7 M progesterone (medroxyprogesterone acetate) (, *p < 0.05) resulted in an upregulation of Sox17 protein, when compared to untreated, estradiol only (■, E) treatment groups. (C,D) Data presented as mean ± SEM, n = 5. Representative Western immunoblot shown. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Knockdown of SOX17 results in reduced expression in endometrial luminal epithelial cells. Transfection of ECC-1 cells with a CRISPR/Cas9 SOX17 knock down plasmid resulted in decreased SOX17 expression (KD1-5) when compared to untransfected control ECC-1 cells (ECC-1) and ECC-1 cells transfected with a control plasmid (CP). Analysed by Western immunoblot, representative blot shown. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664088), licensed under a CC-BY license. Not internally tested by R&D Systems.

Differentiation toward posterior definitive endoderm. (a) Expression of posterior definitive endoderm (DE) marker CDX2 (green), and pluripotency markers OCT4 and NANOG (red). Cells were exposed to LY2090314 or bFGF and induced to differentiate toward PDE on Geltrex. As a control, DE cells were stained against the same antibodies. In the DE there was no CDX2 stain but a clear nuclear localization of pluripotency markers. Scalebar 200 µm. Number of biological replicates n = 5, number of technical replicates n = 2. (b) Expression of posterior definitive endoderm (DE) markers CDX2 (green) and SOX17 (red). Cells were exposed to LY2090314 or bFGF and induced to differentiate toward PDE either on Geltrex or on laminin 111. Number of biological replicates n = 2, number of technical replicates n = 3. (c) In quantitative RT-PCR the bFGF induced higher SOX17 expression than LY2090314. Data expressed as mean ± SD. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35163236), licensed under a CC-BY license. Not internally tested by R&D Systems.

Single-cell RNA sequencing data analysis in human ICMs. A PCA map of human cells from 52 embryos with graded colours indicating FGF4 and SOX17 expression levels in E5 cells and E6-E7 ICM cells (PC1 Score= 28.09%, PC2 score = 11.34%, PC3 score = 6%). b Same PCA plot as in (a) where developmental stages are represented by the indicated colours. c Same PCA plot showing ICMd and ICMp cells (see Supplementary data 8 for the list of cells and their allocation). d Spearman correlation matrix for paired expression of the 308 FGF4-correlated genes in the 258 human ICMd cells. Genes are ordered in a hierarchical tree for similarity (see Supplementary data 9 for detailed map in the vector-based PDF file). e Single cell normalized expression levels of Epi and PrE markers in ICMp (n = 88) and ICMd (FGF4+ (n = 172) and FGF4- (n = 86)) cells (Supplementary data 8); Two-sided Wilcoxon test between FGF4+ and FGF4- ICMd cells (p-values: FGF4p < 2.00 e−16; KLF17p = 5.80 e−11; NANOGp = 5.40 e−09; PRDM14p < 2.00e−16; SOX2p = 6.10e-08; TDGF1p = 2.40 e−13; GATA4p = 0.38; GATA6p = 3.10 e−12; PDGFRAp = 1.40 e−11; SOX17p = 8.00 e−08). For boxplots: The edges of the box represent the 25th and 75th quartiles. The median is represented by the central line. The whiskers extend to 1.5 times the interquartile range (25th to 75th percentile). Cells are plotted individually. Image collected and cropped by CiteAb from the following open publication (//www.nature.com/articles/s41467-022-30858-8), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

, , ,

Applications: , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	AF1924U-100UG
Alexa Fluor 405	AF1924V-100UG
Alexa Fluor 488	AF1924G-100UG
Alexa Fluor 532	AF1924X-100UG
Alexa Fluor 594	AF1924T-100UG
Alexa Fluor 647	AF1924R-100UG
Alexa Fluor 700	AF1924N-100UG
Alexa Fluor 750	AF1924S-100UG
Alexa Fluor Plus 405	AF1924AFP405-100UG
Alexa Fluor Plus 488	AF1924AFP488-100UG
Alexa Fluor Plus 555	AF1924AFP555-100UG
Alexa Fluor Plus 594	AF1924AFP594-100UG
Alexa Fluor Plus 647	AF1924AFP647-100UG
Alexa Fluor Plus 680	AF1924AFP680-100UG
Allophycocyanin	IC1924A
Biotin	BAF1924
CoraFluor 1	AF1924CL1
DyLight 350	AF1924D
DyLight 405	AF1924E
DyLight 488	AF1924K
DyLight 550	AF1924L
DyLight 594	AF1924M
DyLight 650	AF1924W
DyLight 680	AF1924Y
DyLight 755	AF1924Z
FITC	AF1924F
HRP	AF1924H
Janelia Fluor 525	AF1924JF525
Janelia Fluor 549	AF1924I
Janelia Fluor 646	AF1924J
Janelia Fluor 669	AF1924JF669
NL557	NL1924R
PE	AF1924P
PerCP	AF1924C
mFluor Violet 450 SE	AF1924MFV450
mFluor Violet 500 SE	AF1924MFV500
mFluor Violet 610 SE	AF1924MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	IC1924A
Carrier Free	NL1924R
Carrier Free	AF1924-SP

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NB100-1028

AIF-1/Iba1 Antibody

(21 Reviews)

(520 Publications)

0.1 mg

NB100-1028

Reactive microglia (green) in peritumoral area of IC injected human glioblastoma cells in mouse brain. NeuN (red), DAPI (blue). Antibody at 1:250. IHC image submitted by a verified customer review.

AIF-1/Iba1 in canine brain. Primary antibody used at 1 ug/mL and incubated with tissue for 30 minutes at room temperature. IHC-P image submitted by a verified customer review.

Staining in mouse brain using AIF-1/Iba1 Antibody. IHC-P image submitted by a verified customer review.

Brown arrow= AIF-1/Iba1 positive cells, Green arrow= mTOR positive cells, Black arrow= mTOR and AIF-1/Iba1 positive cells. IHC-P image submitted by a verified customer review.

Staining in rat hippocampus using AIF-1/Iba1 Antibody. IHC-Fr image submitted by a verified customer review.

Feline optic nerve microglia were immunolabeled with AIF-1/Iba1 Antibody (green), DAPI (blue). Imaging was performed using an epifluorescence microscope. IHC-Fr image submitted by a verified customer review.

Mouse dorsal root ganglia. AIF-1/Iba1 expression in reactive macrophages. Maximum intensity z-projection from 3D images. IHC image submitted by a verified customer review.

Staining in mouse brain using AIF-1/Iba1 Antibody. IHC image submitted by a verified customer review.

Analysis of LPS injected mouse brain tissue, 20 um thickness, frozen. Primary antibody dilution, 1:200. Secondary antibody CY3. Mounted with DAPI mount. Image from verified customer review.

AIF-1/Iba1 staining of AD mouse model (hippocampus). Image from verified customer review.

Negative Control showing staining of paraffin embedded Human Spleen, with no primary antibody.

(0.1ug/ml) staining of Mouse Lymph node lysate (35ug protein in RIPA buffer). Detected by chemiluminescence.

(1ug/ml) staining of Human Frontal Cortex (A) and (0.1ug/ml) Mouse (B) and Rat (C) Brain lysate (35ug protein in RIPA buffer). Detected by chemiluminescence.

(8ug/ml) staining of paraffin embedded Human Spleen. Heat induced antigen retrieval with citrate buffer Ph 6, HRP-staining.

Immunohistochemistry-Paraffin: Goat Polyclonal AIF-1/Iba1 Antibody [NB100-1028] - AIF-1/Iba1 immunoreactivity in an FFPE section of mouse spleen. NB100-1028 was diluted to 1ug per mL and was left on tissue sections for 30m at room temperature. Secondary was horse anti goat HRP polymer. Image from a verified customer review.

Immunohistochemistry-Paraffin: Goat Polyclonal AIF-1/Iba1 Antibody [NB100-1028] - AIF-1/Iba1 immunoreactivity in an FFPE section of human tonsil. NB100-1028 was diluted to 1ug per mL and was left on tissue sections for 30m at room temperature. Secondary was horse anti goat HRP polymer. Image from a verified customer review.

Immunohistochemistry-Paraffin: Goat Polyclonal AIF-1/Iba1 Antibody [NB100-1028] - AIF-1/Iba1 immunoreactivity in an FFPE section of equine spleen. NB100-1028 was diluted to 1ug per mL and was left on tissue sections for 30m at room temperature. Secondary was equine anti goat HRP polymer. Image from a verified customer review.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Microglia proliferate in non‐APP mice upon peripheral immune challengeA, BIba‐1, Ki67, & DAPI staining in the cortex & hippocampus of wild‐type & Nlrp3−/− (5 & 15 months old). Microglia proliferate upon LPS injection (white arrows). Non‐microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20 μm.CQuantification of microglial proliferation in cortex (left panel) & hippocampus (right panel) (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31359456), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Blood–brain barrier is compromised in old APP mice upon LPS injectionIba‐1 (green) & fibrinogen (red) staining in cortex of 5 & 15 months old of wild‐type & Nlrp3−/− mice. Scale bar: 10 μm.Iba‐1 (green) & fibrinogen (red) staining in cortex of 5 & 15 months old of APP & APP/Nlrp3−/− mice. Scale bar: 10 μm.Fibrinogen integrated density in wild‐type & Nlrp3−/− mice (left panel) & APP & APP/Nlrp3−/− mice (right panel) (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, ***P < 0.001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31359456), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - OTULIN overexpression reduced the number of Iba-1+ microglial cells in the cortical ischemic penumbra in focal cerebral ischemia/reperfusion rats. A Representative images of Iba-1+ microglial cells that were morphologically classified into three types; scale bar = 10 μm. B Iba-1+ microglial cells in the ischemic penumbra & contralateral cortex of each group were detected by confocal immunofluorescence at 24 h, 72 h, & 7 days; scale bar = 75 μm. C The histogram represents the quantitative analysis of mean Iba-1 immunofluorescence intensity in each group (n = 6). Data are presented as the means ± SEMs; ###P < 0.001 versus the tMCAO group; &&&P < 0.001 versus the tMCAO+LV-Scramble group Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29544517), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - ABIN1 knockdown impairs the antineuroinflammatory effect of EA. (a) The concentrations of TNF-alpha , IL-1 beta , & MCP-1 in the peri-infarct cortex were detected using ELISAs at 24 h after reperfusion (n = 5 rats per group). (b) Microglial morphology was observed by conducting immunofluorescence staining for Iba-1 (green) in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (c & d) Quantification of microglial endpoints/cell & process length/cell (∗∗P < 0.01 compared to the MCAO/R group; #P < 0.05 & ##P < 0.01 compared to the MCAO/R + EA group). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33110436), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - a Heatmap of regulated genes related to phagocytosis in the corpus callosum (CC), cerebellum (Cb) & cortex (Cx) at different ages [postnatal day (P)7, 10, 42]. b Heatmap of known priming genes in the CC, Cb & Cx at P7, P10 & P42. Asterisks highlighting genes confirmed on protein level; Representative immunofluorescence pictures of wildtype mice depicting SPP1 [red (c, e)], CLEC7a [white (c, e)] & CD206 [white (d)] expression in IBA-1+ microglia (green) in the CC (c, d), Cx (e left) & Cb (e right) at P7, P10 & P42. Scale bar 50 µm; blue = DAPI; arrow indicating IBA-1+/SPP1+ cell in Cb; N = 3–4 mice per timepoint Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31262353), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Iba1-positive myeloid cells in the ischemic peri-infarct area express cleaved caspase-8 in a pMCAO mouse model of ischemic stroke. Consecutive frozen tissue sections of the ischemic core & the peri-infarct regions from mice after 48 h of permanent middle cerebral artery occlusion (pMCAO) were analyzed by immunohistochemistry with a mouse specific antibody recognizing active caspase-8 when cleaved at Asp 387, or an Iba1 antibody used to detect myeloid cells (a). Double immunofluorescent staining demonstrated the co-localization of cleaved caspase-8 & the myeloid cell marker Iba1 (b). Scale bars represent 850 μm (a) & 300 μm (b) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27566702), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Microglia proliferate in APP/PS1 mice upon peripheral immune challengeA, BIba‐1, Ki67, & MXO4 staining in the cortex & hippocampus of APP & APP/Nlrp3−/− (5 & 15 months old). Microglia proliferate upon LPS injection (white arrows). Non‐microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20 μm.CQuantification of microglial proliferation in cortex (left panel) & hippocampus (right panel) (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31359456), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIF-1/Iba1 Antibody [NB100-1028] - Iba1-positive myeloid cells in the ischemic peri-infarct area express cleaved caspase-8 in a pMCAO mouse model of ischemic stroke. Consecutive frozen tissue sections of the ischemic core & the peri-infarct regions from mice after 48 h of permanent middle cerebral artery occlusion (pMCAO) were analyzed by immunohistochemistry with a mouse specific antibody recognizing active caspase-8 when cleaved at Asp 387, or an Iba1 antibody used to detect myeloid cells (a). Double immunofluorescent staining demonstrated the co-localization of cleaved caspase-8 & the myeloid cell marker Iba1 (b). Scale bars represent 850 μm (a) & 300 μm (b) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27566702), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIF-1/Iba1 Antibody [NB100-1028] - Microglia infiltrate 3×Tg-AD mice brains in a perivascular fashion. Representative hippocampal bright-field × 40 micrographs with × 100 insets. Perivascular infiltration with CD-68+ cells of control (A) & norvaline-treated mice (B) hippocampi. Iba1+ microglia in control (C) & norvaline-treated (D) mice. Measurement of the CD-68 immunopositive surface area (E) & stain intensity (F) with ZEN 2.5 revealed a significant effect of the treatment. (G) The relative intensity of perivascular Iba1+ microglia. Student’s unpaired t-test, n = 20, * p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31540372), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - OTULIN overexpression decreased LPS-induced microglial activation. Pure brain inflammation without neuronal death was induced by IP injection of LPS, & tissues were collected 24 h later. B The mean Iba-1 immunofluorescence intensities in the A LPS(−) group (a), the LPS(+) group (b), the LPS(+)+LV-Scramble group (c), & the LPS (+)+LV-OTULIN group (d) were quantitatively analyzed (n = 6); scale bar = 75 μm. All data are presented as the means ± SEMs; ##P < 0.01 versus the LPS(+) group; &P < 0.05 versus the LPS(+)+LV-Scramble group Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29544517), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Norgestrel significantly abrogates rd10 microglial-induced 661W cell death. (B) Representative fluorescent microscopic image of primary rd10 microglial cells (Iba1; red) in culture with 661W cells (Cone arrestin; green). Hoechst (blue) staining reveals cell nuclei. Scale bar 30μm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0165197), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIF-1/Iba1 Antibody [NB100-1028] - CXCR2+neutrophils are not required for microglial activation. a Representative images of Iba1 immunohistochemistry staining in lumbar spinal cord ventral horns of naïve control, control EAE, & Cxcr2 cKO EAE mice. Quantitative analysis of Iba1+ cell (b) soma area as shown in a. A total of 2338 Iba1+ cells were included in our analyses of soma size (cont naive, 222 Iba1 cells; control EAE, 1236 Iba1+ cells; Cxcr2 cKO EAE, 880 Iba1+ cells). Control naïve, n = 3; control EAE, n = 3; Cxcr2 cKO EAE, n = 3. *p < 0.05, two-tailed unpaired Student’s t test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32019585), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Antibody-based neutralization of Nogo-A mitigates inflammation in the injured retina.a, b Retinal gene expression analysis by qRT-PCR at 3 & 7 days after NMDA injection (0.5 nmol) & antibody treatments (2 μg/eye). Four-six mice were used per condition. 11C7 reduced the expression of major genes involved in inflammation & monocyte activation (*P < 0.05; **P < 0.01; unpaired t-test). In particular, 11C7 injection led to sustained decrease of Tnf transcript & Sprr1a mRNA, the expression of which reflects the severity of neuronal injury response. c The level of TNF alpha trimers was studied by Western Blotting the day after antibody injection. Compared with control IgG (n = 3 mice), 11C7 injections (n = 4 mice) significantly decreased the content of TNF alpha in the vitreous of injured eyes (**P < 0.01, Unpaired t-test). d TNF alpha was mainly observed in Iba1-expressing monocytes by immunofluorescence on retinal flat-mounts. Its signal was much weaker with 11C7 than in control mice. Immunofluorescent staining observations were repeated in three different mice for each antibody treatment. Scale bar in d = 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32029703), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - A20 silencing prevents the anti-inflammatory effects of EA after focal cerebral ischemia reperfusion. Experiments were performed 24 h after reperfusion in the ischemia/reperfusion cortex, n = 5 per group. a TNF-alpha & b IL-1 beta content was measured using ELISA. $$$P < 0.001 vs. the sham group; *P < 0.05, ***P < 0.001 vs. the MCAO group; ###P < 0.001 vs. the MCAO + EA group. The relative mRNA levels of cA1 & dGFAP were measured using RT-qPCR. $$$P < 0.001 vs. the sham group; ***P < 0.001, *P < 0.05 vs. the MCAO group; ###P < 0.001 vs. the MCAO + EA group. The activated morphology of astrocytes & microglia was demonstrated using e GFAP & f Iba-1 immunofluorescence staining (green), respectively. Yellow arrows indicate reactive astrocytes with thick cell bodies, & red arrows indicate hypertrophied activated microglia, noted by their stout, dense appearance with shorter & thicker branched projections. Scale bar = 50 μm Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-016-0731-3), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Norgestrel dampens pro-inflammatory microglial activity in vivo & decreases DAMP release from photoreceptors.(A) Confocal microscopic images of microglia (Iba1; red) & activated microglia (CD68; green) in the central retina from P15-P25 control & Norgestrel-fed rd10 mice. Less microglia including CD68+ microglia were observed at P15 & P20 in Norgestrel-fed mice. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. N = 3 mice per group, n = 8 sections per mouse. (B & C) Quantification of microglial number & CD68-positivity confirmed (B) significantly less microglia at P15 & P20 & (C) significantly less CD68-positivity at P20 with Norgestrel-supplementation. (D) RT-qPCR analysis of mRNA encoding (i) pro-inflammatory (M1) & (ii) & (iii) anti-inflammatory (M2) microglial markers in whole retina from P20-P30 control & Norgestrel-fed rd10 mice. Norgestrel significantly reduced pro-inflammatory & increased anti-inflammatory markers in the retina. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. (E) RT-qPCR analysis of mRNA encoding danger associated molecular patterns (DAMP), (i) high motility box group 1 (HMGB1) & (ii) interleukin-1 alpha (IL-1 alpha ) in whole retina from P20-P30 control & Norgestrel-fed rd10 mice. Norgestrel significantly reduced DAMP expression at P20 & P25 in Norgestrel-fed mice. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.0001). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0165197), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Representative & quantitative immunohistochemistry of the peri-infarct cortex before the bregma (coordinate + 0.58) in normal WT, normal sEH KO, MCAO WT, MCAO sEH KO, MCAO with vehicle treatment & MCAO with AUDA treatment mice (n = 2 for normal groups, n = 5–6 for all groups subjected to MCAO). (A–F, S) The number of CD31-positive endothelial cells significantly increased in the sEH KO before & after MCAO & post-MCAO AUDA-treated mice, while (G–L) Note the increased phosphorylated TrkB (p-TrkB) immunoreactivity that partially co-localized with NeuN-positive neurons, particularly in the sEH KO & AUDA-treated mice (arrows) (T). The percentage of NeuN- & p-TrkB-positive double labeling in the neuronal population was significantly increased compared to that in the corresponding controls. (M–R, U–V) GFAP-positive astrogliosis & Iba1-positive microglial infiltration decreased in post-MCAO sEH KO & AUDA treatment groups relative to that in the corresponding controls. The scale bar represents 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29311641), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - SCF+G-CSF treatment increases the association of Iba1+ microglia/macrophages with senile plaques & enhances uptake of 4G8+ A beta by the Iba1+ microglia/macrophages in the brains of aged APP/PS1 mice. (A) Representative confocal images illustrate the association of Iba1+ cells (green) with 4G8+ A beta plaques (red) in the brains of aged APP/PS1 mice. Blue: Nuclear counterstaining by DAPI. (B) Quantification of the percentage of Iba1+ area in/surrounding the 4G8+ A beta plaques in the brains of aged APP/PS1 mice with or without SCF+G-CSF treatment. (C) Representative 3-dimensional projected images reveal the overlapped (yellow) Iba1+ cells (green) with 4G8+ A beta (red) in the brains of aged APP/PS1 mice. The white arrows indicate the co-expression (yellow) of 4G8+ A beta & Iba1+ cells in the brains of aged APP/PS1 mice. (D & E) Representative orthographic view of z-stack images (12 z-stacks with 1μm intervals) shows the co-expression (yellow) of 4G8+ A beta (red) & Iba1+ cells (green) in the brains of aged APP/PS1 mice. (F) Quantification data present the percentage of 4G8+ A beta volume within the Iba1+ cells in the brains of aged APP/PS1 mice treated with or without SCF+G-CSF. N=4-5. Mean ± SEM. *p<0.05 by Student’s t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33269098), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - The spatial distribution & cell-type location of A20 in the rat brain after I/R. Brain sections of the ischemia/reperfusion cortex of rats in the MCAO + EA group 24 h after reperfusion. Sections were stained with double immunofluorescence labeling (a–d), which showed the upregulated expression of A20 protein (red) in neurons (green, identified using NeuN). e–h Very few of A20-positive (red) astrocytes (green, identified using GFAP) were observed. i–l A20 (red) was barely presented in microglial cells (green, identified using iba-I). Scale bar = 50 μm. 1–3 were the magnified parts from the merged images of d, h, l which more clearly showed the location relationship between A20 & neurons, astrocytes, & microglia, respectively. Scale bar = 25 μm Image collected & cropped by CiteAb from the following publication (//jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-016-0731-3), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Increased perivascular microglia in rTg-DI rats. a–l Brain sections from 3-month-old wild-type (a–c) & rTg-DI (d–f) rats & 12-month wild-type (g–i) & rTg-DI (j–l) rats were labeled with Amylo-Glo to detect fibrillar amyloid (blue), rabbit polyclonal antibody to collagen IV to detect cerebral microvessels (red), & goat polyclonal antibody to Iba-1 to identify microglia (green). Scale bars = 50 μm. m, n Enlarged images of the highlighted regions of panels g & j, respectively. Scale bars = 10 μm. o Quantitation of microglia numbers from wild-type rats (black bars) & rTg-DI rats (gray bars) in different brain regions at 3 & 12 months of age. Data shown are mean ± SD of n = 5–6 rats per group. Compared to wild-type rats the microglia numbers were markedly elevated in rTg-DI rats & increased from 3 to 12 months of age in the measured brain regions. *P < 0.02, **P < 0.01, ***P < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32127016), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Temporal activation of caspase-8 in Iba1-positive myeloid cells. Frozen tissue sections from mice upon pMCAO (n = 9) were analyzed by immunohistochemistry for expression of active caspase-8 or for expression of the microglia/macrophage marker Iba1. The contralateral side to the ischemic area was used an internal control for each animal. A small increase in body size of Iba1 positive cells was detected in the peri-infarct area at 6 h, with larger increase in cell body & numbers of cells expressing Iba1 detected at 24 & 48 h post occlusion (a). Cleaved caspase-8 was detected at low levels at 6 h, but a significant increase in expression levels was observed at 24 & 48 h in the peri-infarct area, but not at the contralateral side (a). Double immunofluorescent staining revealed that increased active caspase-8 expression was localized in Iba1-positive cells (b). Scale bar represent 100 μm in IHC images (a) & 10 μm in IF images (b) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27566702), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - ABIN1 is colocated with A20 & NeuN & Iba-1, respectively, in the peri-infarct cortex. (a) Double immunofluorescence staining for ABIN1 & A20 in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (b) Coimmunoprecipitation of ABIN1 & A20 in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). (c) Double immunofluorescence labeling of ABIN1 (red) & NeuN (green, neurons), Iba-1 (green, microglia), & GFAP (green, astrocytes), respectively (n = 3 rats per group). White arrows show that ABIN1 is colocalized with NeuN & Iba-1, respectively. Scale bar = 50 μm. (d) Comparisons of the percentage of ABIN1+ NeuN+ cells among ABIN1+ cells & ABIN1+ Iba-1+ cells among ABIN1+ cells in the peri-infarct cortex. ∗∗P < 0.01 compared to ABIN1+ NeuN+/ABIN1+. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33110436), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Differential expression analysis in cognitively normal older adults & Plcg2- inactivated mouse model identified inflammation-related pathways. The volcano plot shows significant DEGs (fold change>1.5, FDR-corrected p<0.05) in cognitively normal older adults (CN) with lower (bottom 25%; n=50; low) versus higher (top 25%; n=50; high) expression of PLCG2 (a). Gene-set enrichment analysis was performed on the DEGs (fold change>1.5, FDR-corrected p<0.05) to identify GO terms, including inflammatory response (b). The volcano plot shows significant DEGs derived from the NanoString analysis of the brains of Plcg2 inactivation (PLCG2 inact, n=3) & WT (n=3) mice (1-month male) (c). Immunostaining of microglial markers with IBA1 (green) & P2RY12 (red) & quantification of percent area in brain sections of 1-month-old male WT & PLCG2 inact demonstrate unchanged microglial morphology & area coverage (d). GO gene ontology, BP biological process, DEGs differentially expressed genes, FDR false discovery rate Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35180881), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Norgestrel reduces pro-inflammatory & promotes anti-inflammatory phenotypes in rd10 microglia.(A) RT-qPCR analysis detected similar levels of mRNAs encoding progesterone receptor membrane complexes 1 & 2 (PGRMC1, PGRMC2) & all three membrane progesterone receptor isoforms, alpha , beta & gamma (mPR alpha , mPR beta , mPR gamma ) in rd10 retinal microglial cells. Classical progesterone receptor (PR A/B) was not expressed. Actin, HPRT & GAPDH served as reference genes. (B) Immunolabeling confirmed the presence of these progesterone receptors in primary rd10 microglial cells. Hoechst (blue) staining reveals the cell nuclei. Scale bar represents 10μm. (C) Fluorescent microscopic images of primary rd10 microglia immunolabeled for (C) (i) markers of activation (CD68) & pro-inflammation (M1) (iNOS) & (C) (ii) anti-inflammatory activation (M2) (MRC1); after treatment with 20 μM Norgestrel over 24h. Norgestrel reduced pro-inflammatory & increased anti-inflammatory markers in rd10 microglia. Scale bar 10μm. (D) Quantification of fluorescence intensity of (D) (i) CD68 & iNOS & (D) (ii) MRC1 in primary rd10 microglial cultures following Norgestrel treatment over 24h. N = 8 mice, n = 4 technical replicates per group. (E) Nitrite concentration in the media of rd10 microglia in vitro treated with Norgestrel or vehicle over 24hr. Norgestrel significantly reduced nitrite release from rd10 microglia. N = 8 biological replicates, n = 2 technical replicates per sample. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.001). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0165197), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIF-1/Iba1 Antibody [NB100-1028] - Temporal activation of caspase-8 in Iba1-positive myeloid cells. Frozen tissue sections from mice upon pMCAO (n = 9) were analyzed by immunohistochemistry for expression of active caspase-8 or for expression of the microglia/macrophage marker Iba1. The contralateral side to the ischemic area was used an internal control for each animal. A small increase in body size of Iba1 positive cells was detected in the peri-infarct area at 6 h, with larger increase in cell body & numbers of cells expressing Iba1 detected at 24 & 48 h post occlusion (a). Cleaved caspase-8 was detected at low levels at 6 h, but a significant increase in expression levels was observed at 24 & 48 h in the peri-infarct area, but not at the contralateral side (a). Double immunofluorescent staining revealed that increased active caspase-8 expression was localized in Iba1-positive cells (b). Scale bar represent 100 μm in IHC images (a) & 10 μm in IF images (b) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27566702), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: AIF-1/Iba1 Antibody [NB100-1028] - GM1 effects on brain microglia in vivo, after peripheral administration of LPS. A Schematic representation of in vivo administration of GM1 after LPS-induced systemic inflammation in mice (N = 5 per treatment). B Representative images of Iba-1 stained microglia in the cortex of control animals infused with vehicle (CTRL) or GM1 (CTRL + GM1), & in animals treated with LPS & subsequently infused with vehicle artificial cerebrospinal fluid (LPS) or GM1 (LPS + GM1). C Immunoblot in whole brain lysates & densitometric analysis show LPS-dependent increase in Iba 1 expression. D Cortical & striatal microglia (Iba-1+ cells) cell body area quantified with MetaXpress software. GM1 administration significantly decreases cell body area in the cortex, but not in the striatum. E IL-6 protein levels in whole mouse brain homogenate. aCSF, artificial cerebrospinal fluid. Bars show mean values ± STDEV. Two-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34991625), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - ABIN1 knockdown increases proinflammatory cytokine production & microglial activation. (a-b) Levels of the ABIN1 mRNA & protein were detected 24 h after reperfusion using RT-qPCR & western blot, respectively, to confirm the efficiency of ABIN1 gene knockdown (n = 5 rats per group). (c) ELISA was used to detect the concentrations of TNF-alpha , IL-1 beta & MCP-1 in the peri-infarct cortex at 24 h after reperfusion (n = 5 rats per group). (d) Microglial morphology was observed using immunofluorescence staining for Iba-1 (green) in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (e & f) Quantification of microglia process endpoints & cell & process lengths/cell (∗∗P < 0.01 compared to the sham group; ##P < 0.01 compared to the MCAO/R group). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33110436), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - SCF+G-CSF treatment increases TREM2 expression in the Iba1+ microglia/macrophages surrounding the 6E10+ senile plaques. (A) Representative confocal images of TREM2 (red), 6E10 (purple) & Iba1 (green) triple immunofluorescence staining in the brains of aged APP/PS1 mice. Blue: Nuclear counterstaining by DAPI. (B) Representative orthographic view of z-stack images (12 z-stacks with 1μm intervals) illustrates the location & interaction of TREM2 + cells (red) & 6E10+ A beta plaques (white) in the brains of aged APP/PS1 mice. (C) Quantification data show the percentage of TREM2+ area surrounding the 6E10+ A beta plaques (within 10μm from the border of the A beta plaques) in the brains of aged APP/PS1 mice with or without SCF+G-CSF treatment. (D) Representative orthographic view of z-stack images (12 z-stacks with 1μm intervals) displays the location & interaction of TREM2+/Iba1+ co-expressing cells (yellow) & 6E10+ A beta plaques (white) in the brains of APP/PS1 mice. (E) Quantification data show the percentage of TREM2+/Iba1+ co-expression area in the total of Iba1+ area in the vicinity of 6E10+ A beta plaques in the brains of aged APP/PS1 mice with or without SCF+G-CSF treatment. N=4-5. Mean ± SEM. * p<0.05 by Student’s t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/33269098), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - Antibody-mediated Nogo-A blockade decreases the number of cells expressing TNF alpha without affecting the number of Iba1-positive monocytes.Cells expressing TNF alpha , Iba1, & CD68 were labeled on cryosections by immunofluorescence in 4 mouse retinae, 24 h after control IgG or 11C7 injection. a Retinal sections showed many TNF alpha -containing cells located in the inner retina, between the outer plexiform layer & the ganglion cell layer 1 day after control IgG injection. A majority of cells was positive for Iba1 & exhibited vesicular staining of the lysosomal protein CD68, indicating their active state. b The injection of 11C7 strongly attenuated TNF alpha & CD68 expression & to a lower extent the intensity of Iba1 in macrophages/microglia. c Quantification of the number of cells expressing TNF alpha & Iba1 suggesting potent & specific effects of 11C7 on TNF alpha cytokine reduction. Six sections were examined for each retina. Statistics: one-way ANOVA, Dunnett’s post hoc test, **P < 0.01; ****P < 0.0001. Scale bars = 50 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32029703), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: AIF-1/Iba1 Antibody [NB100-1028] - The presence or absence of TRPV1 influences microglial pathophysiological changes in recurrent febrile seizures mice. (A) Images of the cortex immunostained for the microglial marker Iba-1 (visualized in green FITC) & the lysosomal marker CD68/ED1 (visualized in red Alexa Fluor 594) & DAPI (visualized in blue) 5 days after rFS from wild type (WT) & TRPV1−/− mice (Scale bars: 50 μm). (B,C) Bar graph of quantification of Iba1+(B) & Iba1+ ED1+(C) in 5 days after rFS mice cortex. (D) Representative photomicrographs of different microglial morphological subtypes including resting/ramified (Ram), hypertrophic/intermediate (Inter) & round/ameboid (Amoeboid; Scale bars: 5 μm). (E) Quantification of relative percentage of microglia with different morphologies in the cortex of WT & TRPV1−/− mice (n = 6 per groups, **p < 0.01, ***p < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons). (F–H) Representative Immunoblot bands (F) & densitometric analysis of Iba1 expression (G) & ED1 expression (H) were significantly increased in rFS from WT mice than in TRPV1−/− mice. Data were presented as means ± SEM, n = 6 per groups, *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31680864), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry-Frozen: Goat Polyclonal AIF-1/Iba1 Antibody [NB100-1028] - AD Mouse Model. FxFr:10 µm thickness 1:200. Secondary antibody AF647. Image from a verified customer review.

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Reactivity: Hu, Mu, Rt, Ca, Eq, Fe, Po

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NB600-1384

LC3B Antibody - BSA Free

(19 Reviews)

(459 Publications)

0.1 ml

NB600-1384

LC3B-Antibody---BSA-Free-Immunohistochemistry-NB600-1384-img0044.jpg

<b>Biological Strategies Validation. </b>Analysis of LC3B in treated U87-MG (human glioblastoma astrocytoma) lysates using anti- [Catalog # NB600-1384].

<b>Biological Strategies Validation. </b>LC3B-Antibody---BSA-Free-Western-Blot-NB600-1384-img0040.jpg

<b>Biological Strategies Validation. </b>Human cervical carcinoma (HeLa) and Mouse Neuroblast cells (Neuro2a) were treated with (+) and without (-) 50 uM Chloroquine overnight. Whole cell protein lysates were prepared in 1x Laemmli sample buffer and approximately 10 ug of each lysate (NBP2-49689 and NBP2-49688) was separated on a 4- 20% gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% nonfat milk in TBST. The membrane was probed with 2 ug/mL anti-LC3 (NB600-1384) and 1 ug/mL anti-alpha tubulin (NB100-690) as a loading control, and detected with the appropriate secondary antibodies using NovaLume Pico Chemiluminescence Substrate (NBP2-61915).

<b>Biological Strategies Validation. </b>LC3B Antibody - BSA Free-Immunocytochemistry-Immunofluorescence-NB600-1384-img0045.jpg

Confocal analysis of HeLa cells using Rabbit anti-LC3B antibody (Catalog # NB600-1384, 1:5). An Alexa Fluor 488-conjugated Goat to rabbit IgG was used as secondary antibody (green). Actin filaments were labeled with Alexa Fluor 568 phalloidin (red). DAPI was used to stain the cell nuclei (blue).

<b>Biological Strategies Validation. </b>Immunocytochemical/Immunofluorescent staining of treated U373-MG cells using the HRP conjugate of anti- (Catalog # NB600-1384). The nuclei were stained with DAPI.

<b>Biological Strategies Validation. </b>Staining of treated U373-MG (human glioblastoma) cells using anti- [Catalog # NB600-1384].

Analysis of U87MG glioma xenografts using anti- [Catalog # NB600-1384]. Image from verified customer review.

<b>Biological Strategies Validation. </b>LC3B staining in treated U87-MG cultured & subcutaneous tumors.

LC3B staining in glioblastoma multiform tissue.

Lane view shows a specific band for LC3B in 0.5 mg/ml of Neuro2A lysate at a molecular weight of approximately 15 kDa. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

LC3B-Antibody---BSA-Free-Knockdown-Validated-NB600-1384-img0034.jpg

<b>Biological Strategies Validation. </b>LC3B-Antibody---BSA-Free-Electron-Microscopy-NB600-1384-img0038.jpg

Examples of immunohistochemical stainings; (A) LC3B low, (B) LC3B high, (C) p62 low (any cellular compartment), (D) p62 cytoplasmic low, (E) p62 cytoplasmic/dot like high, nuclear low, (F) p62 cytoplasmic/dot like low, nuclear high, (G) HMGB1 low, (H) HMGB1 high. Objective magnification, 40×.

Simple Western: LC3B Antibody - BSA Free [NB600-1384] - Autolysosome imaging after growth factor deprivation captured the autophagy induction in iPSC-derived astrocytes. (A) Schematic procedures of autophagy induction assay. (B) Representative images of cells and autolysosomes on day 2 with or without growth factor deprivation. (C) Fluorescence counts per well of 96-well plate at 16 h after initiation of assay. Astrocytes were treated with control, starvation and starvation with 10 nM bafilomycin A1 (BafA1) condition. The data were analysed by one-way ANOVA (F [3, 12] = 21.19, p < 0.0001), followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ****p < 0.001, ns, not significant. Data represent mean +- SD (n = 4, from different experimental replicates). (D) Astrocytes were treated with control, starvation and starvation with 10 nM bafilomycin A1 (BafA1) condition. Cells were lysed and subjected to immunoblot analysis at 16 h after initiation of assay. M.W., molecular-weight size marker. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/38509731 ), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Autophagy associated protein immunoreactivity in HIV-infected brain tissue. (A) Representative images from five randomly selected fields of cells each examined in duplicate frontal lobe white matter sections for the indicated subject groups. The indicated proteins were labeled red & microglia with the cell-type-specific marker Iba1 (green). Blue staining indicates cell nuclei. Arrow heads indicate examples of higher Iba1 immunoreactivity whereas arrows indicate more focal (punctal) vs. diffuse (filamentous) patterns of autophagy associated protein expression. Scale bar = 10 μm. (B) Quantification of relative Iba1 immunoreactivity from (A). F(3,20) = 6.450, p = 0.0031; ∗p < 0.05 when compared to all other subject groups. Error bars show the SEM for the average values of 2–6 regions from each subject group across the six autophagy associated proteins examined. (C) Quantification of the indicated autophagy associated protein relative immunoreactivity from (A). Beclin 1: F(3,12) = 11.29, p = 0.0008; LC3B: F(3,12) = 1.994, p = 0.1687; APG7/ATG7: F(3,12) = 84.20, p = < 0.0001; ATG5: F(3,12) = 6.218, p = 0.0086; p62/SQSTM1: F(3,12) = 87.04, p = < 0.0001; LAMP1: F(3,12) = 8.317, p = 0.0029. ∗p < 0.05 when compared to HIV-negative; #p < 0.05 when compared to HIV-positive; & Ωp < 0.05 when compared to HIV-positive/NCI subjects. Error bars show the SEM for four regions from each subject group. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/Article/10.3389/fmicb.2015.00653/abstract), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Fucoxanthin failed to suppress oxidative stress & activate autophagy in Nrf2−/− mice following TBI.Fucoxanthin treatment had no effect on change the level of MDA & the activity of GPx (A) & the expression of Beclin-1, LC3 & p62 (B) in Nrf2−/− mice compared to the vehicle-treated group. n = 6 per group. @p > 0.05 versus TBI + vehicle group. beta -actin was used as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28429775), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Differences in neuronal autophagy & dendrite varicosity following HIV-1 Tat protein & morphine treatment. (A) Representative images of neurons transfected with a fluorescent reporter plasmid to monitor autophagic flux at 8 h following the indicated treatments. GFP (green) & GFP + mRFP (yellow) fluorescence are observed prior to the fusion of autophagosomes with lysosomes whereas only mRFP (red) fluorescence is present in post-fusion autolysosomes. DIC, differential interference contrast microscopy image. DAPI (blue) staining indicates cell nuclei. (B) Quantification of autolysosomes (red puncta) from (A). F(3,13) = 8.756, p = 0.0019; ∗p < 0.05 when compared to all other groups. (C) Western blotting analysis of the indicated autophagy associated protein levels at 24 h following the indicated treatments. GAPDH was used as a loading control. Blots are representative of three independent experiments. (D) Quantification of dendrite beading from (A). F(3,77) = 6.429, p = 0.0006; ∗p < 0.05 when compared to control cells. Error bars show the SEM. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/Article/10.3389/fmicb.2015.00653/abstract), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

HMGB1 regulated autophagy in thyroid cancer cells (a) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA & control shRNA & then starved by HBSS for 2 h. & LC3-I/II level was assayed by Western blot; (b) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA & control shRNA & then pre-treated for 1 h with pepstatin A (PA, 10 μM) & E64D (10 μM) as indicated. Cells were subsequently treated for 3 h with HBSS in continuous presence or absence PA/E64D inhibitors. LC3-I/II, Beclin1 & p62 levels were assayed by Western blot; (c) Ultrastructural features in FTC-133/TPC-1 cells transfected with HMGB1 shRNA & control shRNA after a 3-h treatment of HBSS. More autophagosomes were seen in control shRNA plus HBSS-treated cells than in cells treated with HMGB1 shRNA plus HBSS. Arrows indicated autophagosomes Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31331356), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Fucoxanthin activated autophagy after TBI.(A) Representative images of immunofluorescence for LC3 surrounding the injured cortex. LC3 punctate dots were observed in the cytoplasm by immunofluorescent staining of LC3 (red). Neuron cells & nuclei are labeled with NeuN (green) & DAPI (blue), respectively. Magnification: 40 x. Scale bar: 50 mm. (B) Mice brain tissues were collected 1 day after TBI in different groups, & the expression of LC3, Beclin-1 & p62 was measured by western blot. Fucoxanthin treatment significantly increased the level of LC3-II & Beclin-1 while decreasing the level of p62 after TBI. (C) 3-MA (400 nM) was injected i.c.v. 30 min before TBI. Mice were then subjected to TBI & treatment of fucoxanthin 30 min after TBI. Pretreatment with 3-MA significantly attenuated fucoxanthin-induced activation of autophagy & suppression of apoptosis & oxidative stress in the ipsilateral cortex. Data are presented as mean ± SEM, n = 6 per group; **p < 0.01, ***p < 0.001 versus sham group; #p < 0.05, ##p < 0.01 versus TBI + vehicle group; &&p < 0.01, &&&p < 0.001 versus TBI + fucoxanthin group. beta -actin was used as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28429775), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Fucoxanthin protected primary cultured neurons from TBI.(A) Primary cortical neurons were subjected to scratch injury & then treated with 5, 10 or 20 μM fucoxanthin or DMSO for 1 day. The LDH release assay was used to evaluate cell viability. The percentage of survival cells significantly decreased after TBI compared to the control group. Fucoxanthin treatment significantly increased survival cells after TBI. (B) Fucoxanthin repressed the production of ROS in primary cultured cells after TBI. Cells were subjected to scratch injury & subsequently treated with 100 μM edaravone or 5, 10 or 20 μM fucoxanthin or DMSO for 1 day. Then cells were incubated with DCFH-DA & subjected to fluorescence spectrophotometer analysis. The intracellular ROS was significantly increased after TBI compared to the sham group, & administration of edaravone or fucoxanthin significantly repressed ROS production as compared to the TBI + DMSO group. (C) Fucoxanthin inhibited apoptosis & activated autophagy in primary cultured neurons. Primary cortical neurons were subjected to scratch injury & then treated with 5, 10 or 20 μM fucoxanthin or DMSO for 1 day, the expression of cleaved caspase-3, Beclin-1, LC3 & p62 was measured by western blot. Fucoxanthin significantly decreased the expression of cleaved caspase-3 & p62 while increased the expression of Beclin-1 & LC3-II. Data are presented as mean ± SEM, n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001 versus control group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus TBI + DMSO group; @p > 0.05 versus TBI + DMSO group. beta -actin was used as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28429775), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

8-Cl-Ado-induces autophagic cell killing. (A) Western blot analysis of beclin1 & ATG7 levels in MCF-7 cells transfected with either a pool of control siRNA (siCONT), siRNA targeting the expression of the beclin1 gene (siBECN1), or targeting the expression of the ATG7 gene (siATG7). Immunoblot analysis of LC3B lipidation & PARP cleavage were assessed as markers of autophagosome formation & apoptosis, respectively. GAPDH was used as loading control. Flow cytometric analysis of cells transfected with siCONT, solid bars, siBECN1, hatched bars, or siATG7, checkered bars, treated with 10 μM 8-Cl-Ado & stained with (B) annexin V & PI, as well as (C) acridine orange. Effect of autophagy on 8-Cl-Ado-inhibiton of clonogenic survival. Cells transfected with (D) siCONT, ○, or siBECN1, ●, & with (E) siCONT, ○, or siATG7, ●, were treated with the indicated doses of 8-Cl-Ado for 3 days, washed with PBS, & cultured in fresh medium for 10 days. Colonies of >50 cells were counted under a dissecting microscope. Image collected & cropped by CiteAb from the following publication (//jhoonline.biomedcentral.com/articles/10.1186/1756-8722-7-23), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB600-1384] - Assesment of functional basal autophagy in vitro(A) Immunoblotting of LC3B & p62 upon pharmacological autophagy inhibition. A dose-dependent increase of LC3B & p62 was observed with increasing concentrations of chloroquine. (B) Immunofluorescence of LC3B & p62 upon pharmacological autophagy inhibition. I. In CC cell lines HT-29 & II. LoVo a dose-dependent increase of LC3B & p62 was observed with increasing concentrations of chloroquine. (C) Electron microscopy of CC cell lines pharmacological autophagy inhibition. I-III. In CC cell line HT-29 a dose-dependent increase of cytoplasmic vesicles was observed with increasing concentrations of CQ. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28903368), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Phosphorylation of AKT & mTOR decreases after treatment with gemcitabine (GCB)A LM7, CCH-OS-D, & K7M3 osteosarcoma cells were treated with 1μM GCB & cell lysates were analyzed by immunoblotting with specific antibodies for AKT, mTOR, p-AKT, & p-mTOR. beta -actin was used as a loading control. Densitometry was performed using ImageJ. B. & C. Following treatment with GCB, lysates were analyzed using the Human Phospho Kinase Antibody Array. The phosphorylation levels of AKT, mTOR, PRAS40, p70S6K, JNK, AMPK, & ERK were evaluated. Densitometry was performed using Image J. Values were graphed as relative fold change. Means ± standard deviation of three independent experiments are shown. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.20308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Autophagy decreases during striatal development. (A) Representative Western blot images for LC3B, p62, DARPP32, & actin. Quantification of (B) LC3B-ii relative to actin, (C) LC3B-ii relative to LC3B-i, (D) p62 relative to actin normalized to P120 values. Data analyzed with one-way ANOVA; (B) Age: F(4,21) = 6.526, p = 0.0014; (C) Age: F(4,22) = 3.797, p = 0.0171; (D) Age: F(4,15) = 3.762, p = 0.0260. *p < 0.05, **p < 0.01, n = 4–6 mice/age. (E,F) Representative images of DARPP32 stained striatal neurons & RFP fluorescence from mice aged P10 & P28. Dashed lines indicate cell body outlines. (G) Quantification of number of LC3 puncta/cell. Unpaired, two-tailed t-test, t4 = 4.392, p = 0.0118. N = 3 mice/age, 20–50 cells were analyzed per mouse. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32296308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: LC3B Antibody - BSA Free [NB600-1384] - Examples of immunohistochemical stainings; (A) LC3B low, (B) LC3B high, (C) p62 low (any cellular compartment), (D) p62 cytoplasmic low, (E) p62 cytoplasmic/dot like high, nuclear low, (F) p62 cytoplasmic/dot like low, nuclear high, (G) HMGB1 low, (H) HMGB1 high. Objective magnification, 40×. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30134604), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Validation of modulations in autophagy-related markers after SIRT6 knockdown in melanoma cells(A) RT-qPCR analysis was performed to validate the mRNA levels in key altered genes after SIRT6 knockdown A375 cells. Results are expressed as SIRT6 knockdown (shSIRT6) mRNA levels compared to nonsense control (shNS). Data are mean ±SE of three biological replicates, with statistical significance denoted as *P≤ 0.05. (B) Western blot analyses of various autophagy markers including LC3 I & II, BECN1, SQSTM1, ATG3, ATG7, ATG10 & GAA using cell lysates of shNS & shSIRT6 in A375 melanoma cells. beta -actin was used as loading control. The figure shows representative western blot images from 2-3 experiments. Image collected & cropped by CiteAb from the following publication (//www.genesandcancer.com/lookup/doi/10.18632/genesandcancer.153), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Fucoxanthin activated autophagy after TBI.(A) Representative images of immunofluorescence for LC3 surrounding the injured cortex. LC3 punctate dots were observed in the cytoplasm by immunofluorescent staining of LC3 (red). Neuron cells & nuclei are labeled with NeuN (green) & DAPI (blue), respectively. Magnification: 40 x. Scale bar: 50 mm. (B) Mice brain tissues were collected 1 day after TBI in different groups, & the expression of LC3, Beclin-1 & p62 was measured by western blot. Fucoxanthin treatment significantly increased the level of LC3-II & Beclin-1 while decreasing the level of p62 after TBI. (C) 3-MA (400 nM) was injected i.c.v. 30 min before TBI. Mice were then subjected to TBI & treatment of fucoxanthin 30 min after TBI. Pretreatment with 3-MA significantly attenuated fucoxanthin-induced activation of autophagy & suppression of apoptosis & oxidative stress in the ipsilateral cortex. Data are presented as mean ± SEM, n = 6 per group; **p < 0.01, ***p < 0.001 versus sham group; #p < 0.05, ##p < 0.01 versus TBI + vehicle group; &&p < 0.01, &&&p < 0.001 versus TBI + fucoxanthin group. beta -actin was used as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28429775), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB600-1384] - Fucoxanthin activated autophagy after TBI.(A) Representative images of immunofluorescence for LC3 surrounding the injured cortex. LC3 punctate dots were observed in the cytoplasm by immunofluorescent staining of LC3 (red). Neuron cells & nuclei are labeled with NeuN (green) & DAPI (blue), respectively. Magnification: 40 x. Scale bar: 50 mm. (B) Mice brain tissues were collected 1 day after TBI in different groups, & the expression of LC3, Beclin-1 & p62 was measured by western blot. Fucoxanthin treatment significantly increased the level of LC3-II & Beclin-1 while decreasing the level of p62 after TBI. (C) 3-MA (400 nM) was injected i.c.v. 30 min before TBI. Mice were then subjected to TBI & treatment of fucoxanthin 30 min after TBI. Pretreatment with 3-MA significantly attenuated fucoxanthin-induced activation of autophagy & suppression of apoptosis & oxidative stress in the ipsilateral cortex. Data are presented as mean ± SEM, n = 6 per group; **p < 0.01, ***p < 0.001 versus sham group; #p < 0.05, ##p < 0.01 versus TBI + vehicle group; &&p < 0.01, &&&p < 0.001 versus TBI + fucoxanthin group. beta -actin was used as a loading control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28429775), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Striatal autophagic flux decreases from P10 to P28. (A) Schematic of coronal brain section showing dissection boundaries for ex vivo experiments. (B) Quantification of LC3B-ii relative to actin for every vehicle-only slice showed in Figures 4, 5. Unpaired, two-tailed t-test, t52 = 5.824, ****p < 0.0001. (C) Representative Western blot images of actin, DARPP32 & LC3B in slices obtained from P10 or P28 mice, incubated with BafA1 (100 nM, 3 h) or vehicle (Veh; DMSO, 0.1%). (D) LC3B-ii relative to actin, normalized to vehicle condition at each age. P10: unpaired, two-tailed t-test, t25 = 5.113, ****p < 0.0001; P28: unpaired, two-tailed t-test, t10 = 0.6228, p = 0.5473. P10: Veh: n = 16 slices, BafA1 n = 11 slices from 4 to 6 mice. P28: Veh: n = 6 slices, BafA1 n = 6 slices from three mice. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32296308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - The activated HeyL-aromatase axis promotes autophagic survival of PCSCs. (A) Gene signatures associated with autophagy-associated signaling pathways identified by GSEA in HeyLHigh samples vs. HeyLLow samples from the TCGA-PRAD dataset & in HeyL knockdown cells vs. ctrl cells. (B, C) Western blot analysis of Beclin1, LC3, & CD44 in LNCaP (B) & PC3 (C) cells after the indicated treatments. (D) Flow cytometry analysis of the CD44+/CD24− subpopulation in PC3 cells treated as indicated. (E, F) mRNA (E) & protein (F) levels of stemness-associated genes & LC3 in PC3 cells after the indicated treatment (one-way ANOVA). (G, H) mRNA & protein levels of stemness-associated genes & autophagy-related genes in PC3 cells treated with ICI182780 (G) & siER alpha (H) (t-test). (I, J) Cleaved caspase3 levels in LNCaP & PC3 cells after the indicated treatments (one-way ANOVA). (K, L) Cleaved caspase3 levels in PC3 cells treated with ICI182780 (K) or transfected with siER alpha (L) (t-test). Bic, bicalutamide; CQ, chloroquine. The data are presented as the mean ± SD values (n=3). *p < 0.05 vs. ctrl. #p < 0.05 vs. OE-HeyL, i_HeyL or OE-CYP19A1. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35096586), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Treatment with gemcitabine (GCB) induces autophagy in osteosarcoma cellsA. Following 48 hours of treatment with 1μM GCB, LM7, CCH-OS-D, & K7M3 cells were stained with the lysosomotropic agent acridine orange (1 μg/ml) for 15 minutes & then visualized under a confocal microscope. Treatment with GCB led to the formation of acidic vesicular organelles (AVOs –red/orange), as shown in the representative images. B. Flow cytometry was used to measure & quantify AVO formation in untreated (left) & treated (right) osteosarcoma cells. C. Cells were treated with 1μM GCB for 48 hours, & after fixation, cells were stained with anti-LC3 antibody & analyzed by fluorescence confocal microscopy. GCB increases the number of green punctae in the treated group as compared to the untreated. D. Expression of BECN, LC3, & p62 was analyzed using Western blot analysis. Cells were treated with GCB for 48 hours, lysed & extracted protein was analyzed using specific antibodies. beta -actin was used as a loading control. Autophagy induction is demonstrated by an increase in the conversion of LC3I to LC3II, BECN expression & a decrease in p62 expression in the treated group as compared with the untreated. E. Treatment of OS cells with GCB (1μM, 48 hours), led to an increase in the formation of autophagic vacuoles (shown with red arrows) measured by transmission electron microscopy. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.20308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - RAR alpha but not RAR beta & RAR gamma agonists induce autophagic flux in SKBR3 cells. (a) SKBR3 cells were treated with 1μM of RAR alpha , RAR beta or RAR gamma agonists for 2 days in the presence & absence of BafA during the last 2 h, before subjection to immunofluorescence for LC3B. (b) Quantification of LC3B puncta from the experiment described in (a). Three independent experiments were quantified as described in Schläfli et al.41 (c) Autophagic flux was determined from the immunofluorescence analysis shown in (a). N=3, Student's t-test & **P<0.01. (d) Long-lived protein degradation assay of control or cells treated with 1 μM of RAR alpha , RAR beta , RAR gamma agonists in the presence or absence of 200 nM BafA. Radioactivity was determined by liquid scintillation counting of four independent experiments. Data are shown as BafA-sensitive proteolysis. (e) Western blot analysis for VE-cadherin, beta -catenin & LC3B of SKBR3 cells treated as in (a). (f) LC3B-II levels on western blot were normalized to GAPDH & quantified from at least five independent experiments using the ImageJ software (NIH, Bethesda, MD, USA). LC3B-II levels of control treated cells were arbitrarily set to 100%. Mann–Whitney U-test: *P<0.05, **P<0.01 & ****P<0.0001 Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2015236), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - ROS/AMPK/mTOR pathway was required for HMGB1-mediated autophagy regulating NIS expression (a) FTC-133/TPC-1 cells transfected with HMGB1 shRNA & control shRNA transfection were starved by HBSS for 3 h & then treated with rapamycin (1 μM) for 12 h. ROS production was assessed by measuring the fluorescent intensity of DCF on a fluorescent plate reader. Incremental production of ROS was expressed as a percentage of control. Rap, rapamycin. (n = 3, *P < 0.01, **P > 0.01); (b) FTC-133/TPC-1 cells transfected with HMGB1 shRNA & control shRNA were starved by HBSS for 3 h & then treated with rapamycin (1 μM) for 12 h. Flow cytometry was performed for measuring the ROS level by a DCFH-DA probe in indicated cells. Rap, rapamycin. (n = 3, *P < 0.01, **P > 0.01); (c) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA & control shRNA & then starved by HBSS for 3 h. ATP levels were detected by ATP Assay Kit (n = 3, *P > 0.01); (d) FTC-133/TPC-1 cells were pretreated with NAC (2 mM) & then starved by HBSS for 3 h. LC3-I/II, NIS, p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K & p70S6K levels were assayed by Western blot; (e) FTC-133/TPC-1 cells transfected with HMGB1 shRNA & control shRNA were starved by HBSS for 3 h & then treated with rapamycin (1 μM) for 12 h. LC3-I/II, NIS, p-mTOR & mTOR levels were assayed by Western blot. Rap, rapamycin Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31331356), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Serum starvation induces autophagy & apoptosis in endothelial cells.A Evaluation by Hoechst 33342 & propidium iodide (HO/PI) staining of apoptosis & necrosis in HUVECs exposed to normal medium (N) or serum-starved (SS) for 4 h. P values obtained by unpaired t test. n = 10 for each condition. Scale bar = 25 μm. B Quantification of caspase-3 activity in HUVECs exposed to N or SS HUVECs for 1, 2, 3, & 4 h. P values obtained by one-way ANOVA. n = 4 for each condition. C Immunoblot & densitometric analysis of PARP, LC3, & SQSTM1/p62 in HUVECs exposed to N or SS for 1, 2, or 4 h. beta -actin (ACTB) was used as a loading control. P values obtained by unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001). n = 3 for each condition. All values are expressed as the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35149669), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - 4-phenylbutyric acid (4-PBA) alleviated zearalenone (ZEN)-induced ER stress, autophagy, & downregulation of CYP3A4 & UGT1A. Before treatment with ZEN (20 & 40 μg/mL), cells were pretreated with 1 mM 4-PBA for 6 h. mRNA & protein expression were determined by quantitative real-time PCR (qRT-PCR) & Western blot analysis, respectively. (A) Cells were treated with ZEN for 24 h after pretreatment with 4-PBA & cell viability was measured by the trypan blue dye exclusion test. (B) Phosphorylation of eIF2 alpha was evaluated in cells treated with ZEN for 2 h. The protein expression of (C) LC3-II/LC3-I & (D) CYP3A4 was determined in cells treated with ZEN for 24 h. (E) UGT1A mRNA levels were determined in cells treated with ZEN for 16 h. Data represent mean ± SEM of three independent experiments. * indicates significant difference vs. the control (* p < 0.05, ** p < 0.01, & *** p < 0.001). # indicates significant difference between groups (p < 0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31861425), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Bmi‐1‐RING1B & autophagy are negatively related to SA‐PCH. (A) Kyoto Encyclopedia of Genes & Genomes (KEGG) analysis was conducted using differentially expressed genes (DEGs) in young (14‐week‐old) & old (18‐month‐old) mouse hearts. (B) Gene set enrichment analysis (GSEA) on autophagy signal pathway & ubiquitin‐mediated proteolysis. (C) Genes Correlation analysis using 12‐ & 18‐ month mouse hearts RNAseq count data. (D) Gene Ontology (GO) analysis & GSEA were processed using genes that were highly related to Bmi‐1. (E) Western blots of cardiac extracts of WT young (8‐week‐old) or aging (20‐month‐old) mice showing Bmi‐1, RING1B, LC3BII, Ang II, GATA4, ANP, BNP, NF‐ kappa B‐p65 & p‐p65 (Ser536), I kappa B‐ alpha & p‐I kappa B‐ alpha (Ser32); beta ‐actin was the loading control. (F) Protein levels relative to beta ‐actin were assessed by densitometric analysis. Six mice per group were used for experiments. Values are mean ± SEM of six determinations per group, **p < .01, ***p < .001 compared with the WT young mice, unpaired t‐test for bar graphs Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35390228), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - HMGB1-mediated autophagy regulated NIS expression & iodide uptake (a). FTC-133/TPC-1 cells were pretreated with 3-MA (10 mM) treatment for 1 h & then starved by HBSS for 3 h. LC3-I/II & NIS levels were assayed by Western blot; (b&c) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA & control shRNA & then starved by HBSS for 3 h. LC3-I/II & NIS levels were assayed by Western blot. & NIS mRNA was assayed by qRT-PCR (n = 3, *P > 0.01, **P > 0.01); (d) Dynamic uptaking: FTC-133/TPC-1 cells were transfected with HMGB1 shRNA & control shRNA & starved by HBSS for 3 h. Indicated cells were cultured with 175 KBq 131I for 5, l0, 20, 30, 60, 90 & 120 min. The uptake of 131I in indicated cells was detected by a gamma counter; (e) Radionuclide uptaking: FTC-133/TPC-1 cells were transfected with HMGB1 shRNA & control shRNA in the presence or absence of 3-MA (10 mM) treatment for 1 h & then starved by HBSS for 3 h. After 1-h incubating with 131I, the uptake of 131I in indicated cells was detected by a gamma counter (n = 3, *P < 0.01, **P > 0.01); (f) FTC-133/TPC-1 cells transfected with HMGB1 shRNA & control shRNA were starved by HBSS for 3 h & then treated with rapamycin (1 μM) for 12 h. After 1-h incubating with 131I, the uptake of 131I in indicated cells was detected by a gamma counter. Rap, rapamycin. (n = 3, *P < 0.01, **P > 0.01) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31331356), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: LC3B Antibody - BSA Free [NB600-1384] - ROS was sufficient for inducing HMGB1 translocation & enhancing autophagy (a) FTC-133/TPC-1 cells were pretreated with the antioxidant (NAC, 2 mM) for 1 h & then starved by HBSS for 3 h. ROS production was assessed by measuring the fluorescent intensity of DCF on a fluorescent plate reader. Incremental production of ROS was expressed as a percentage of control (n = 3, *P < 0.01, **P < 0.01). UT, untreated group; (b) Antioxidant & SOD1 RNAi regulated starvation-induced autophagy as measured by LC3-II expression. FTC-133/TPC-1 cells were pretreated with NAC (2 mM) for 1 h or SOD1 RNAi for 48 h, & then starved by HBSS for 3 h. LC3-I/II level was assayed by Western blot. (c) Antioxidant & SOD1 RNAi regulated starvation-induced HMGB1 translocation. FTC-133/TPC-1 cells were pretreated with NAC (2 mM) for 1 h or SOD1 RNAi for 48 h, & then starved by HBSS for 3 h. & the expression of nuclear/cytosolic HMGB1 was assayed by Western blot. Fibrillarin was a nuclear fraction control & tubulin a cytoplasmic fraction control Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31331356), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Autophagy was induced under conditions of LH & inhibited by CQ & 3-MA in HCC cells.(A–C). SMMC-7721 & HepG2 cells were directly incubated under normal or LH conditions for 8 h or incubated under LH conditions in the presence or absence of CQ or 3-MA for 8 h. (A). The cells were transfected with GFP-tagged LC3 & observed with fluorescence microscopy. Arrows show the punctate GFP-LC3 fluorescence in the cytoplasm. (B). The number of punctate GFP-LC3 in each SMMC-7721 or HepG2 cell was counted, & at least 100 cells were included for each group. The data represent the mean ± SD based on three independent experiments. **p < 0.01. (C). The whole-cell lysates were subjected to western blot analysis as described in the ‘Materials & methods'section. The experiments were repeated at least three times. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep05382), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Combination treatment of valproic acid (VPA) & doxorubicin (DOX) synergistically augmented the autophagy of HepG2 cells. (A) Acridine orange (AO) staining was used to detect acidic vesicles in HepG2 cells at the indicated concentration of VPA & DOX monotherapies & combination treatment after incubation for 48 h. Images were taken using fluorescence inverted microscopy. Red color represents acidic vesicle & green color represents non-acidic vesicle. Scale bar represents 200 μm; (B) The viability of HepG2 cells was analyzed after 48-h incubation in the indicated experimental condition by using EZ-Cytox assay; (C) Percentages (%) of AO-positive cells were counted in different fields (containing at least 40 cells per field); (D) LC3 I & II protein levels were analyzed using Western blotting. Actin was used as the loading control. The intensity of LC3B-II bands was quantified by scanning densitometry program ImageJ & normalized to that of actin (right panel). Three independent experiments were performed & results reported as the mean ± standard deviation (SD). ** p < 0.01, *** p < 0.001 compared with the control group. Image collected & cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/18/5/1048), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Metformin treatment increases cisplatin effects; (A) MDA-MB-231 & Ca Ski cells were either left untreated or treated with metformin or cisplatin alone or metformin in combination with cisplatin for 15 days. At the end of the treatment, cells were processed to obtain whole cell lysates. MAP1LC3B, pPRKAA2Thr172, PRKAA2 & CASP3 (cleaved) expression was determined by Western blot. ACTB was used as loading control. Densitometric analysis of the gels was performed as described under Materials & Methods; (B) MDA-MB-231 & Ca Ski cells were either left untreated or treated with metformin or cisplatin alone or metformin in combination with cisplatin for 15 days. Ammonia level in the culture medium was measured as indicated in the Material & Methods section. # Significantly different from treatment with metformin alone. All experiments in this figure were repeated three times. * Significantly different from control untreated cells. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30646605), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Autophagy & the proteasome regulate CagA stability. (A) Wild-type (WT) & autophagy-deficient (Atg5−/−) MEFs were infected with a CagA+ vacA− isogenic mutant strain (MOI 100) for 8 hours using a gentamycin protection assay. The autophagy marker LC3-II was used to confirm that Atg5−/− MEFs are autophagy-deficient. CagA protein levels were measured by Western blotting using beta -actin as loading control. Graph shows fold change of CagA normalized to beta -actin relative to WT MEFs (mean + SEM; n = 4). (B) AGS cells were infected with a CagA+ vacA− isogenic mutant strain (MOI 50) for 19 hours using a gentamycin protection assay. MG132 (5 μM) was added during the 14-hour low-dose gentamycin incubation. DMSO was used as a vehicle control. CagA protein levels were measured by Western blotting using beta -actin as loading control. Graph shows fold change of CagA normalized to beta -actin relative to vehicle control (mean + SEM; n = 6). (C) AGS cells were infected with a CagA+ vacA− isogenic mutant strain (MOI 50) for 24 hours using a gentamycin protection assay. Lactacystin (10 μM) was added during the 19-hour low-dose gentamycin incubation. Water was used as a vehicle control. CagA protein levels were measured by Western blotting using beta -actin as loading control. Graph shows fold change of CagA normalized to beta -actin relative to vehicle control (mean + SEM; n = 5). Relevant gel bands were cropped from the original blots. Dotted line indicates slicing of two regions together from the same blot. Statistical analysis was performed using Student’s t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30631092), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Vps34 activity is required for maintaining LC3B-ii levels at P10 & mTOR inhibition increases p-Beclin-1 & LC3B-ii levels at P28. (A) Representative Western blot images for actin, DARPP32 & LC3B-i & -ii in striatal slices obtained from P10 or P28 mice, treated with SAR405 (1 μM) or vehicle (Veh; DMSO, 0.1%). (B,C) Quantification of LC3B-ii relative to actin, normalized to vehicle condition at each age. (B) P10: unpaired, two-tailed t-test, t11 = 2.985, p = 0.0124; P28: unpaired, two-tailed t-test, t24 = 1.807, p = 0.0922. P10: Veh: n = 6 slices, SAR405 n = 7 slices from 3 to 4 mice/age. (C) P28: Veh: n = 9 slices, Baf n = 7 slices from 3 to 4 mice/age. *p < 0.05. (D) Representative Western blot images for actin, DARPP32, p-Beclin-1 Ser14 & LC3B-i & -ii in striatal slices from P28 mice, treated with Torin-1 (5 μM) or vehicle (Veh; DMSO, 0.1%). Quantification of (E) LC3B-ii & (F) p-Beclin S14 relative to actin, normalized to vehicle. Data analyzed with unpaired, two-tailed t-test; (E) LC3B-ii/actin, t24 = 2.853, p = 0.0088. Veh: n = 19 slices, Baf n = 7 slices from 5 to 7 mice. *p < 0.05. (F) p-Beclin S14/actin, t22 = 3.337, p = 0.0030. **p < 0.01, Veh: n = 18 slices, Torin-1 n = 6 slices from 3 to 5 mice. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32296308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: LC3B Antibody - BSA Free [NB600-1384] - After stimulation with the NOD1 ligand, macrophages increased their expression of autophagy proteins Atg9, LC3 & IRGM. Alveolar macrophages (AMs), monocyte-derived macrophages (MDMs) & monocytes (MNs) were incubated in the presence of 5 μg/ml of Tri-DAP for 24 h. Cells were pre-incubated with Rip2/p38 inhibitor SB203580 (SB) for 30 min prior to Tri-DAP stimulation to block NOD1-mediated responses, as indicated. Atg9 & LC3 proteins were measured in the cytosolic fractions by western blot analysis (A). The fold increases relative to the unstimulated cells were calculated after being normalized to tubulin, mean ± SE is depicted, n = 3 (B, C). The up-regulation of IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system & delta delta CT method for relative quantification. The fold changes in gene expression relative to the unstimulated cells of at least 6 subjects are depicted; bold lines indicate medians, *p < 0.05 (D). IRGM protein was measured in the cytosolic fractions of three subjects by western blot, & the fold increases relative to the unstimulated cells are indicated after being normalized to tubulin; mean ± SE is depicted, n = 3 (E, F). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25253572), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB600-1384] - The effect of H19 siRNA on autophagy in OGD/R model. A) Immunofluorescence of LC3II in different conditions. B) Immunofluorescence of Beclin1 in different conditions. C) Immunofluorescence of P62 in different conditions. D) The change of lncRNA H19 expression level induced by H19 siRNA. E. Western blot of LC3II, LC3I, Beclin1, & P62 in different groups. F, G, & H) Statistical analysis of the expression of LC3II, Beclin1 & P62, respectively. N, normal group; OGD/R, OGD 8 hr & reperfusion 24 hr; OGD/R + H19 siRNA & OGD/R + N.C, transfecting the cells with H19 siRNA & normal control siRNA, respectively, before OGD/R treatment. Bar represents the mean value ± SD. *p<0.05 vs. normal group, #p<0.05 vs. OGD/R group, &p<0.05 vs. OGD/R + H19 siRNA group. White arrows indicate the co-localized positive cells. Image collected & cropped by CiteAb from the following publication (//www.aginganddisease.org/EN/10.14336/AD.2016.0530), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Inhibition of autophagy impacts on ATRA-induced epithelial differentiation of SKBR3 cells. (a) SKBR3 cells were treated for 4 days with 1 μM ATRA, increasing concentrations of chloroquine or a combination thereof as indicated before western blot analysis for VE-cadherin, beta -catenin, LC3B & GAPDH. (b) Quantification of beta -catenin & VE-cadherin western blots as one is shown in (a). Raw values were normalized to GAPDH & levels of ATRA-treated control transduced cells were arbitrary set to 100% as signals were not always detectable in vehicle-treated control cells. Bars & s.d. representative of five independent experiments. (c) SKBR3 control & ATG7-depleted cells were subjected to 1 μM ATRA for 4 days & expression levels of VE-cadherin, beta -catenin, ATG7 & GAPDH were determined by western blot. (d) Quantification of western blots for beta -catenin & VE-cadherin. Normalization was performed as described in (b) Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2015236), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Autophagy controls the cleavage of perlecan to LG3 fragments in apoptotic exosome-like vesicles (ApoExos).A Serum anti-LG3 IgG titers in allografted mice after 3 weeks of intravenous injections with vehicle (Ctrl) or ApoExos produced by serum-starved mECs treated with vehicle (ApoExo V), bafilomycin A1 (20 nM, ApoExo Baf), or ZVAD-FMK (50 μM, ApoExo ZVAD). P values were obtained by one-tailed unpaired t test. ns (nonsignificant). n = 7 mice for V & Baf injections, & n = 9 mice for V & ZVAD-FMK injections. B Representation of the abundance of perlecan peptides in ApoExos from serum-starved mECs treated with vehicle (V) or bafilomycin A1 (20 nM, Baf). Fold enrichment of perlecan peptide intensity in ApoExos from bafilomycin-treated cells compared to ApoExos from vehicle-treated cells. C Representative immunoblots & densitometric analysis of perlecan & LG3 fragments in ApoExos purified from HUVECs serum starved (SS) for 4 h in the presence of V or Baf. Ponceau red was used as a loading control. P values were obtained by the unpaired t test. n = 3 for each condition. *P < 0.05; **P < 0.01; ***P < 0.001. All values are expressed as the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35149669), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Induction of autophagic flux in OE19 upon Lapatinib treatment. (a) LC3B flux was assessed comparing control & BafilomycinA (BafA)-treated (200 nM, 2 h) OE19 upon Lapatinib treatment (120 nM, 24 h). LC3 band intensities were quantified using ImageJ software. Total protein was used as a loading control, & Phospho-Her2 for Lapatinib treatment (n = 3). (b) LC3B flux was calculated from data in (a) as follows: BafA+-BafA− values for each condition. (c) FACS analysis of mCherry-EGFP-LC3B-expressing OE19 cells upon induction or blockade of autophagy with indication of the chosen cut-off value for high respectively low autophagic flux. (d) Quantification of the FACS analysis showing % of cells with high autophagic flux (n = 3). Cells were treated as in (a). The error bars represent SD, statistical significance was determined by Mann–Whitney U test: * p ≤ 0.05, ** p ≤ 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30297650), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Autophagy regulates the loading of LG3 on apoptotic exosome‐like vesicles (ApoExos).A Representative immunoblots & densitometric analysis of LC3, ATG7, phospho-AKT1, & total AKT1 from serum-starved (SS) HUVECs transfected with siCtrl or siATG7 or exposed to vehicle (V), wortmannin (100 nM; Wort), or bafilomycin A1 (20 nM, Baf). beta -actin (ACTB) was used as a loading control. n = 3 for each condition. B HO/PI staining of apoptosis in SS HUVECs transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. n = 3–5 for each condition. ns (nonsignificant). C Immunoblots & densitometric analysis of cleaved caspase-3 in SS HUVECs transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. beta -actin (ACTB) was used as a loading control. n = 3–5 for each condition. D Small particle flow cytometric quantifications of CMFDA+ AnnexinV+ ApoExos detected in the supernatant of HUVECs serum starved (SS) for 4 h & transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. n = 3 for each condition. E Representative immunoblots & densitometric analysis of LG3, 20S proteasome, LAMP2, & synthenin-1 in ApoExos purified from HUVECs serum starved (SS) for 4 h & transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. Ponceau red was used as a loading control. n = 3–5 for each condition. P values were obtained by the unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001). All values are expressed as the mean ± SEM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35149669), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB600-1384] - Treatment with gemcitabine (GCB) induces autophagy in osteosarcoma cellsA. Following 48 hours of treatment with 1μM GCB, LM7, CCH-OS-D, & K7M3 cells were stained with the lysosomotropic agent acridine orange (1 μg/ml) for 15 minutes & then visualized under a confocal microscope. Treatment with GCB led to the formation of acidic vesicular organelles (AVOs –red/orange), as shown in the representative images. B. Flow cytometry was used to measure & quantify AVO formation in untreated (left) & treated (right) osteosarcoma cells. C. Cells were treated with 1μM GCB for 48 hours, & after fixation, cells were stained with anti-LC3 antibody & analyzed by fluorescence confocal microscopy. GCB increases the number of green punctae in the treated group as compared to the untreated. D. Expression of BECN, LC3, & p62 was analyzed using Western blot analysis. Cells were treated with GCB for 48 hours, lysed & extracted protein was analyzed using specific antibodies. beta -actin was used as a loading control. Autophagy induction is demonstrated by an increase in the conversion of LC3I to LC3II, BECN expression & a decrease in p62 expression in the treated group as compared with the untreated. E. Treatment of OS cells with GCB (1μM, 48 hours), led to an increase in the formation of autophagic vacuoles (shown with red arrows) measured by transmission electron microscopy. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.20308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Activation of autophagy by MG132 is associated with AGR2 decline. a The expression change of AGR2 in A549 cells was estimated by western blotting analysis. A549 cells were incubated with CHX (2 μg/ml) for 2 h prior to MG132 treatment. GAPDH served as a loading control. b AGR2 ubiquitination was detected by immunoprecipitation with anti-AGR2 antibody & immunoblotting with anti-Ub antibody. Similar results were obtained with three independent experiments. c Immunofluorescence microscopy for analysis of punctate pattern of LC3 localization in A549 cells transiently transfected with the GFP-LC3-vector for 24 h & treated with MG132 for additional 12 or 24 h, & LC3 punctate (right panel) was calculated in A549 cells. **p < 0.01 compared with the control. Scale bar = 20 μm. d The levels of AGR2 & LC3B were measured by western blotting with MG132 treatment at indicated time. e Analysis of LC3B & AGR2 accumulation in A549 cells by fluorescent staining. Scale bar = 10 μm Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30647455), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - VPA reduces autophagy at P28. (A) Schematic of VPA administration. (B) Representative Western blot images for LC3B, p62, p-rpS6 S240/244, DARPP32, & actin. (B–G) Quantification of (B–D) p-rpS6 S240/244 relative to actin, (B,E,F) LC3B-ii relative to actin (B,G,H) p62 & (B,I,J) DARPP32 normalized to VPA untreated (veh, vehicle) mice at P10 & P28, respectively. Data analyzed with unpaired, two-tailed t-test; (C) p-rpS6 S240/244/actin at P10, ns; (D) p-rpS6 S240/244/actin at P28, t11 = 2.882, p = 0.0149 (E) LC3B-ii/actin at P10, ns; (F) LC3B-ii/actin at P28, t10 = 2.231, p = 0.047; (G) p62 at P10, ns; (H) p62 at P28, t10 = 3.752, p = 0.0038; (I) DARPP32 at P10, ns, & (J) DARPP32 at P10, ns, *p < 0.05, **p < 0.01, ns not significant, n = 4–7 mice/treatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32296308), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: LC3B Antibody - BSA Free [NB600-1384] - In vitro alpha S seeds induce endo-lysosome-free & endo-lysosome-associated alpha S inclusions in H4/V1S-SV2 cells. (a) Gel analysis of in vitro prepared alpha S seeds. Recombinant alpha S solution subjected to 7 days of shaking was ultracentrifuged (110,000 × g, 20 min), & the resultant supernatant & pellet were resolved by SDS-PAGE & western blotting with anti-alpha S antibody (Synuclein-1, 610787, BD Biosciences). A second aliquot without shaking was included as a control. (b) Electron micrographs of alpha S seeds before & after sonication. Scale bar: 100 nm. Different alpha S polymorphs are shown in close-ups. (c) H4/V1S-SV2 cells treated with sonicated alpha S seeds for 2 days were subjected to immunocytochemistry with primary antibody against LAMP1 followed by DAPI counterstain to demonstrate the association between endo-lysosomes & seeded alpha S inclusions. The bottom two rows are magnified images of frames A & B in the second row. Empty triangles & solid arrowheads denote the endo-lysosome-free & endo-lysosome-associated alpha S inclusions, respectively. Scale bar: 10 µm (top two rows) & 5 µm (bottom two rows). (d) The treated cells were further subjected to immunocytochemical staining of LAMP1 (Alexa fluor 405) & LC3 (Alexa fluor 568) to demonstrate the association between autophagosomes, endo-lysosomes, & seeded alpha S inclusions. Scale bar: 5 µm (top row) & 2 µm (bottom row). Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-08149-w), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - CA1 Tg mice undergo mitochondrial stress. a WB analysis of extracts from 6-mo Wt & Tg mice CA1 (Wt, n = 6; Tg, n = 7), using antibodies against SIRT3, SOD2 & acetylated SOD2. b WB analysis of extracts from 12-mo Wt & Tg mice hippocampi (Wt, n = 5; Tg, n = 5). Data are presented as mean ± SEM (**P < 0.01; ***P < 0.001). WB analysis of extracts from 6-mo Wt & Tg mice CA1 (Wt, n = 6; Tg, n = 5) (c), & from 12-mo Wt & Tg mice hippocampi (Wt, n = 5; Tg, n = 5) (d) for PINK1 & LC-3. Data are presented as mean ± SEM, *P < 0.05. WB analysis of extracts from 6-mo WT & Tg mice CA1 (WT, n = 5; Tg, n = 6) (e), & from 12-mo WT & Tg mice hippocampi (WT, n = 5; Tg, n = 5) (f) for TFAM. Data are presented as mean ± SEM (*P < 0.05), **P < 0.01). Actin was used as loading control Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32131898), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Effects of exogenous HMGB1 on ATRA-induced differentiationA. Viability of NB4 cells that were treated with exogenous HMGB1 (10, 20 & 50 μg/ml) for 6-72 h was determined by the CCK-8 assay. Viability of control cells (DMSO) was set as 100%. (n=3, *P <0.05 versus control group). B. LDH released by NB4 cells that were treated with HMGB1 (10 μg/ml) for 6-48 h was detected by LDH assay kit & expressed as percentage of control (n=3, *P>0.05 versus untreated group). C. Morphological features of NB4 cells that were treated with ATRA (1 μM) or HMGB1 (1 μg/ml) was determined by Wright-Giemsa staining & visualized by light microscopy (100X magnification). D. Differentiation of NB4 cells treated with ATRA (1 μM) or HMGB1 (10 μg/ml) for 6-48 h was examined by NBT reduction & surface CD11b expression in comparison to the control (DMSO) (n=3, *P<0.05 versus ATRA group). E. Expression of PML-RAR alpha , P62 & LC3-I/II in NB4 cells treated with ATRA (1 μM) or HMGB1 (10 μg/ml) for 48 h was assayed by western blotting & co-immunoprecipitation in comparison to control (DMSO). Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.15432), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Zearalenone (ZEN) induced endoplasmic reticulum (ER) stress & autophagy in HepG2 cells. mRNA level & protein expression were determined by quantitative real-time polymerase chain reaction (qRT-PCR) & Western blot analysis, respectively. (A) The protein expression level of GRP78 was measured in cells treated with ZEN for 1 h. (B) The phosphorylation of eIF2 alpha was evaluated in cells treated with ZEN for 2 h. (C) CHOP mRNA levels, (D) Beclin1 mRNA levels, & (E) LC3-II mRNA levels were determined in cells treated with ZEN for 4 h. (F) The protein expression level of LC3-II/LC3-I was examined in cells treated with ZEN for 24 h. Data represent mean ± SEM of three independent experiments. * indicates significant difference vs. the control (** p < 0.01, & *** p < 0.001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31861425), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - ATRA induces autophagy in SKBR3 cells. (a) SKBR3 & MDA-MB-453 breast cancer cells were treated either with 0.1 or 1 μM ATRA for 2 & 4 days, respectively. beta -Catenin & LC3B levels were measured by western blotting. GAPDH was used as a loading control. (b) LC3B-II quantification from at least three independent experiments. LC3B-II expression was normalized to GAPDH & to vehicle-treated SKBR3 cells. (c) beta -Catenin staining of SKBR3 & MDA-MB453 cells. Cells were control or ATRA (1 μM) treated for 2 days. beta -Catenin FITC (fluorescein isothiocyanate) & nuclear DAPI (4',6-diamidino-2-phenylindole) staining as analyzed by confocal microscopy are shown. (d) Quantification of endogenous LC3B puncta. LC3B in SKBR3 & MDA-MB453 cells treated with 1 μM ATRA for 2 days. (e) LC3B-II western blotting & quantification of the autophagic activation upon treatment with 1 μM ATRA for 2 days in the presence or absence of BafA for 2 h at 200 nM. LC3B-II levels were normalized to GAPDH & to vehicle-treated SKBR3 cells. Standard deviations for five independent experiments are shown. (f) Long-lived protein degradation assay of SKBR3 & MDA-MB453 cells treated as in (e). Radioactivity was determined by liquid scintillation counting of at least three independent experiments. Absolute proteolysis is shown. (g) Long-lived protein degradation assay of SKBR3 treated as in (e) & including treatment with the macroautophagy-specific inhibitor 3-MA (10 mM). Analysis as in (f). Mann–Whitney U-test: *P<0.05, **P<0.01, ***P<0.001 & ****P<0.0001 Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2015236), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - Bmi‐1 deficiency aggravates cardiac hypertrophy & decreases autophagic flux. (A) Representative micrographs of paraffin‐embedded heart ventricular wall sections of 6‐week‐old Bmi‐1–/– mice & WT mice stained for haematoxylin‐eosin (H&E), wheat germ agglutinin (WGA) & Masson's trichrome (Masson) staining. (B) Heart weight relative to body weight (HW/BW) & heart weight relative to tibia length (HW/TL). (C) The wall thickness of interventricular septum. (D) Myocyte cross‐sectional area from Bmi‐1–/– mice relative to WT mice. (E) Collagenous fibre area from Bmi‐1–/– mice relative to WT mice was counted with anterior wall staining for Masson. (F) Anp, Bnp, beta ‐MHC, c‐TnI & GATA4 mRNA levels in hearts by real‐time RT‐PCR, calculated as ratio to beta ‐actin mRNA & expressed relative to WT. (G) Western blots of cardiac tissue extracts & mouse embryonic cardiomyocytes (MECs) showing ANP, BNP, GATA4, LC3B & p16; beta ‐actin was the loading control. (H) Protein levels relative to beta ‐actin were assessed by densitometric analysis. (I) Representative micrographs of paraffin‐embedded heart sections immunohistochemical staining for ANP, BNP & GATA4. (J) The percentage of cells positive for ANP, BNP, GATA4 or positive area relative to total cells or area. (K) Representative micrographs of fluorescence with autophagy lentivirus transfection, with DAPI for nuclei. (L) Percentage of positive GFP dots or mRFP dots relative to total cells. (M) The number of autophagosomes & autolysosomes per cell. Six mice per group were used for experiments. Cell experiments were performed with three biological repetitions per group. Values are mean ± SEM from six determinations per group, *p < .05, **p < .01, ***p < .001 compared with WT group, unpaired Student's t‐test Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35390228), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - 8-Cl-Ado-induces autophagy. Immunoblot analysis of (A) phospho-ULK (Ser555) levels & (B) LC3B-I lipidation to form LC3B-II in MCF-7 & BT-474 cells treated with 10 μM of 8-Cl-Ado for the indicated times. (C) Fluorescent microscopy of MCF-7 & BT-474 cells transiently transfected with GFP-LC3B & treated with 10 μM of 8-Cl-Ado for 12-hours to assess aggregation of LC3B. (D) Immunoblot analysis of p62 levels in MCF-7 & BT-474 cells treated with 10 μM of 8-Cl-Ado for the indicated times. The normalized ratio of p62 to GAPDH relative to the untreated control is indicated beneath each lane. (E) Fluorescent microscopic imaging of autolysosomes. MCF-7 cells were untreated or treated for 2-days with 8-Cl-Ado or rapamycin. Cells were stained with MDC, blue, to visualize AVO & Syto 61 (DNA), red, for nuclei counterstaining. (F) Representative flow cytometery histograms of AVO stained with acridine orange in MCF-7 cells untreated or treated for 3 days with 50 nM rapamycin or 10 μM 8-Cl-Ado. Baf was added before 30 min prior to staining to neutralize AVO staining. (G) Quantification triplicate experiments of MCF-7 & BT-474 cells treated & analyzed as in E. Image collected & cropped by CiteAb from the following publication (//jhoonline.biomedcentral.com/articles/10.1186/1756-8722-7-23), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: LC3B Antibody - BSA Free [NB600-1384] - PB induces autophagy but blocks autophagosome trafficking & autophagosome-lysosome fusion in Hep3B cells.(A) Hep3B cells, HepG2 cells, & primary mouse hepatocytes treated w/ increasing concentrations of PB (6–2000 μg/mL) for 24 h; viable cells quantified using the MTT assay. Data are presented as mean ± SD from at least 3 independent experiments. Bars = SD. *P < 0.05, **P < 0.01, ***P < 0.001, control versus PB-treated cells. (B) Hep3B cells treated for 6, 24 & 48 h w/ indicated concentrations of PB. Levels of protein expression analysed by WB using antibodies against LC3B, p62/SQSTM1, ATG12, BECN1 & Actin. Images cropped from different blots run under the same experimental conditions. The original blots attached as Supplementary Figure 3A. (C) Hep3B cells transfected w/ GFP-LC3B treated w/ 240 μg/mL PB for 24 h & then observed under a confocal microscope. (D) Hep3B cells treated for 24 h w/ indicated concentrations of PB. Levels of protein expression analysed by WB using antibodies against phosphorylated & total mTOR, 4EBP1, & p70S6K1 as well as actin. Images cropped from different blots run under the same experimental conditions. The original blots attached as Supplementary Figure 3B. (E) Hep3B cells serum-starved overnight, & incubated w/ or w/out PB (240 μg/mL) for 6 h before stimulating w/ 20 ng/mL EGF for 20 min. EGFR protein levels analysed by confocal microscope. (F) Hep3B cells treated PB for 12, 24 or 48 h w/ indicated concentrations. Levels of protein expression analysed by WB using antibodies against EGFR & Actin. Images cropped from different blots run under the same experimental conditions in each panel. The original blots attached as Supplementary Figure 3C. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/srep41437), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Ba, Bv, Ca, Pm, Ye, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Ba, Bv, Ca, Pm, Ye, Ze

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB600-1384AF350
Alexa Fluor 405	NB600-1384AF405
Alexa Fluor 488	NB600-1384AF488
Alexa Fluor 532	NB600-1384AF532
Alexa Fluor 594	NB600-1384AF594
Alexa Fluor 647	NB600-1384AF647
Alexa Fluor 700	NB600-1384AF700
Alexa Fluor 750	NB600-1384AF750
Alexa Fluor Plus 405	NB600-1384AFP405
Alexa Fluor Plus 488	NB600-1384AFP488
Alexa Fluor Plus 555	NB600-1384AFP555
Alexa Fluor Plus 594	NB600-1384AFP594
Alexa Fluor Plus 647	NB600-1384AFP647
Alexa Fluor Plus 680	NB600-1384AFP680
Allophycocyanin	NB600-1384APC
Biotin	NB600-1384B
CoraFluor 1	NB600-1384CL1
DyLight 350	NB600-1384UV
DyLight 405	NB600-1384V
DyLight 488	NB600-1384G
DyLight 550	NB600-1384R
DyLight 594	NB600-1384DL594
DyLight 650	NB600-1384C
DyLight 680	NB600-1384FR
DyLight 755	NB600-1384IR
FITC	NB600-1384F
HRP	NB600-1384H
Janelia Fluor 525	NB600-1384JF525
Janelia Fluor 549	NB600-1384JF549
Janelia Fluor 646	NB600-1384JF646
Janelia Fluor 669	NB600-1384JF669
PE	NB600-1384PE
PerCP	NB600-1384PCP
mFluor Violet 450 SE	NB600-1384MFV450
mFluor Violet 500 SE	NB600-1384MFV500
mFluor Violet 610 SE	NB600-1384MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB600-1384SS

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NB300-221

GAPDH Antibody (1D4) - BSA Free

(19 Reviews)

(455 Publications)

0.1 ml

NB300-221

Analysis of GAPDH in human nucleus pulposus cell culture (40ug/lane) using anti-GAPDH antibody. LI-COR Odyssey imaging system was used for detection. Western blot image submitted by a verified customer review.

Analysis of of HeLa cells stained with GAPDH antibody, dilution 1:100 (Green). DAPI staining of nuclear DNA (Blue). The GAPDH antibody produces strong cytoplasmic staining of healthy cells.

Analysis of cell line lysates probed with mouse mAb to GAPDH (theoretical molecular weight: 36 kDa), NB300-221, dilution 1:2,000: [1] protein standard, [2] HEK293, [3] HeLa, [4] SH-SY5Y, [5] COS1, [6] NIH-3T3, and [7] C6 cells. The GAPDH antibody reveals a single band at ~37 kDa in all cell lines. GAPDH is a house keeping protein, the level of which is relatively unaffected by most experimental manipulations, and, as a result, this antibody has been widely used as a western blotting loading control.

Human eye cell lysate. Antibody used at 1:500. WB image submitted by a verified customer review.

Analysis of GAPDH in chicken pectoralis major using anti-GAPDH antibody. The primary antibody was used at a dilution of 1:4000, incubated for 1 hour at room temperature. Western blot image submitted by a verified customer review.

<b>Biological Strategies Validation. </b>GAPDH-Antibody-1D4-Western-Blot-NB300-221-img0015.jpg

<b>Biological Strategies Validation. </b>GAPDH-Antibody-1D4-Western-Blot-NB300-221-img0014.jpg

SH-SY5Y cells stained with GAPDH antibody NB300-221 (green). Nuclear DNA is stained with Hoechst dye (blue).

Lane view shows a specific band for GAPDH using 0.05 mg/mL Jurkat and MCF-7 cell lysates and antibody at 1:25. Electropherogram image of corresponding Simple Western lanes view at WES molecular weight of 40 kDa. Image reported by internal validation.

Knockdown of Ift88 or Kif3a in NP cells does not affect hyperosmotic upregulation of TonEBP. (a) TonEBP/Nfat5 mRNA levels in NP cells with Ift88 knockdown (n ≥ 5). (b) Western blot image showing increased TonEBP expression in response to hyperosmolarity (550 mOsm/kg H2O) independently of Ift88 knockdown. (c) Densitometry analyses of TonEBP with Ift88 knockdown (n ≥ 4). (d) TonEBP/Nfat5 mRNA levels in NP cells with Kif3a knockdown (n ≥ 3). (e) Western blot image showing that hyperosmotic induction of TonEBP is maintained after Kif3a knockdown. (f) Densitometry analyses of TonEBP after Kif3a knockdown (n ≥ 4). Data are represented as scatter plots (mean ± SEM). ns = not significant. One-way ANOVA or Kruskal-Wallis test with Sidak’s or Dunn’s multiple comparison test was used based on the distribution of the data to determine statistical significance. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Fig. S1-1 for un-cropped Western blot images. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664118), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - OPN induces EndoMT. (E) VE-cadherin, Tie1, Tie2, CD31, alpha -SMA, & fibronectin levels in HUVECs pre-incubated 16 h with 2% FBS-containing medium & then treated 24 h with PBS or 0.3 μg/ml of OPN. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29435158), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - miR-21-5p delivery by H-EV treatment promote NSCLC cell proliferation, survival & mobility by targeting PTEN, PDCD4 & RECK. a–d, western blot detecting PTEN, PDCD4 & RECK protein expression level in A549 or H23 cells after different treatment as described in Fig. 4. e–k, cell functional assay as described in Fig. 1. A549 or H23 cells with or without PTEN, PDCD4 or RECK gene overexpression (O/E) were tested. Wild type, non-treated A549 or H23 cells were used as negative control (NC). Bar in K indicates 20 μm. Tukey’s test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30736829), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Expression of CTGF & TGF‐ beta 1 in WT & MMP‐13 KO mice treated without & with NDMA. (A) Real‐time PCR analysis for the quantitative expression of CTGF, TGF‐ beta 1, alpha ‐SMA & collagen type 1 mRNA in WT & MMP‐13 KO mice. The data are mean ± S.D. of six samples per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice; *P < 0.05 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; #P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice. (B) Semiquantitative RT‐PCR for the expression of CTGF, TGF‐ beta 1, alpha ‐SMA & type 1 collagen mRNA in WT & MMP‐13 KO mice treated without & with NDMA. GAPDH was used as a loading control. The data are representative of six samples in each group. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28782260), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Hydrogen sulfide donors leads to inhibition of phosphate transport. Cells were cultured in a normal or calcific environment exposed to AP72 (20 μmol·L−1) for 5 days. (a) Phosphate content was determined using QuantiChrom quantitative colorimetric assay. (b) Pit1 & (c) Pit2 western blotting was performed & normalized to GAPDH. Data shown are means ± SEM of five independent experiments. *P < .05, significantly different as indicated; ns, not significant Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31017307), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Overexpression of NICD increases the total Fmod protein contents in BMDMs & LPS prevents the cleavage of Fmod in a dose dependent manner. (A,B) WB shows the expression of total Fmod in Apoe−/− & Notch1+/−;Apoe−/− BMDMs & quantification of three replicates as determined by Image J. (C,D) WB showing the total Fmod protein content in WT BMDMs 48 h post transfection with empty or NICD plasmids & the quantification of the immunoblots. (E,F) qRT-PCR showing the panel of M1 & M2 genes dysregulated with human recombinant FMOD (400 ng/ml for 24 h). (G) Co-immunoprecipitation showing contents of Tgf-beta 2, Fmod & NICD proteins pulled down with Fmod antibody from the WT & Notch1+/− peritoneal macrophages. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31142802), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Lactate dehydrogenase (LDH) assay & the Western blot of kidney injury molecule (KIM)-1 & N-cadherin in rat mesangial cells (RMCs). Cells were treated with bicalutamide (Bic) at concentrations of 30 & 60 μM at 24 h. (a) LDH release was increased by Bic (n = 3, * p < 0.05). (b) A representative blot of protein expressions of KIM-1 & N-cadherin. GAPDH was used as an internal control. (c) Quantitative data of Western blotting of KIM-1(n = 3, * p < 0.05). (d) Quantitative data of Western blotting of N-cadherin (n = 3, * p < 0.05). When RMCs were treated with Bic, N-cadherin dose-dependently decreased, however KIM-1 was significantly induced in the group treated with 60 μM. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32403414), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Immediate early gene (IEG) expression in RICH2 KO mice. IEG expression was analyzed in nuclear protein lysate from amygdala & normalized to GAPDH levels. No significant difference was detected in the expression of (A) activity-regulated cytoskeleton-associated protein (ARC; Unpaired t-test, n = 3, p = 0.077), & (B) c-FOS (Unpaired t-test, n = 3, p = 0.8784). A significant difference was detected in the expression of (C) cyclic AMP response element-binding protein (CREB; Unpaired t-test, n = 3, p = 0.002). The amount of phosphorylated CREB (pCREB) was unchanged (Unpaired t-test, n = 3, p = 0.1822). Exemplary Western blot bands are shown. Histone H3 was used a control for the presence of nuclear proteins in the lysate. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/article/10.3389/fnmol.2017.00180/full), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Relative Myonectin/CTRP15 content.Relative Myonectin/CTRP15 content was examined in the rat diaphragm muscle of the male lean zucker (LZR) & obese zucker rats (OZR). Exercised animals were trained on a motorized treadmill for 9 wk. Control animals were exposed to the similar environment (positioned next to the treadmill) but were not exercised. (A) Shows representative western blots for Myonectin & GAPDH. (B) The data are expressed in arbitrary units with values normalized to mean control value within phenotype. Image collected & cropped by CiteAb from the following publication (//peerj.com/articles/605), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - FerrylHb induces HO-1 & ferritin in EC. Confluent HUVECs grown on 6-well plates were exposed to heme, Hb, metHb, & ferrylHb (100 μmol/L heme or as indicated in complete medium containing 15% of FBS). After 4 hours of incubation total RNA was isolated & HO-1 mRNA level was measured by quantitative RT-PCR (panels (a) & (d)). For protein expression, HUVECs were solubilized after 8 hours of treatment. HO-1 & ferritin H & L expression was detected by Western blot ((b) & (e)) or with ELISA (c). Immunoblots were reprobed with GAPDH & are representative of three independent experiments. Results are shown as mean ± S.D. (n = 3) from one representative experiment of three. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23766856), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - microRNA-200c regulates MARCKS.(A) The effect of miR-200c transfection on mRNA levels of predicted target genes in Wm266.4 melanoma cells was assessed using GAPDH as a control. Lower panel: immunoblot of MARCKS expression. (B) Brightfield imaging & morphological quantification of Wm266.4 cells transfected with siRNAs targeting miR-200c-responsive genes. (C) MARCKS knock-down by four individual siRNAs leads to rounded cell shape in Wm266.4 cells. (D) Levels of MARCKS mRNA in melanoma cell lines & normal melanocytes, measured by qPCR & normalized to levels of GAPDH. Error bars represent ± SEM; unpaired t-test *p<0.05 **p<0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20957176), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Dose-dependent effects of LPS/IFN-gamma on Tgf-beta expression are associated with changes in the cleaved Fmod fragments. (A) Representative WB image showing the contents of NICD, Mmp8, total Fmod, cleaved Fmod, Tgf-beta 2 & Gapdh in macrophages in response to increasing dose of LPS (0, 5, 10, 25, 50 or 100 ng/ml) for 24 h. (B–F) Quantitation of immunoblots for NICD, Mmp8, total Fmod, cleaved Fmod, Tgf-beta 2 respectively (average of three replicates shown after normalizing the intensity with Gapdh. (WB = Western blot). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31142802), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - OPN induces EndoMT. (C) mRNA levels of VE-cadherin, Tie1, Tie2, CD31, alpha -SMA, & fibronectin in HUVECs pre-incubated 16 h with 2% FBS-containing medium & then treated 15 h with PBS or 0.3 μg/ml of OPN. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29435158), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - miR-200a associated elongation is associated with decreased actomyosin contractility.(A) The effect of miR-200a transfection on mRNA levels was measured for predicted target genes in Wm266.4 melanoma cells using GAPDH as a control. (B) Transfection of Wm266.4 cells with miR-200a leads to decreased pMLC levels. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20957176), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Regulation of cell migration-associated proteins by NCKU-21 in A549 & CL1-5 cells.Protein expressions of matrix metalloproteinase-2 (MMP-2) & MMP-9 were analyzed in A549 (A) & CL1-5 (B) cells treated with NCKU-21 for 24 hr. * P < 0.05 & ** P < 0.01, compared to the control group (without NCKU-21 treatment). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28945763), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - TNF alpha negatively regulates EC differentiation: the effect of TNF alpha on VEGF-A-stimulated differentiation of MSCs into ECs was examined. TNF alpha R & VEGFR-2 mRNA expression was analyzed by RT-PCR (n = 3) (a). Sox18 protein levels were measured by Western blot analysis (n = 3) (b). Expression of EC markers was determined by FACS analysis, & a representative grid is shown (n = 3-4) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay (n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗p < 0.05 versus naïve MSCs & #p < 0.05 MSCs treated with VEGF-A versus VEGF-A plus TNF alpha cotreatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26106428), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - RICH2 is expressed at glutamatergic synapses in the amygdala. (A) Expression of Rich2 was detected on mRNA level using a PCR approach in amygdala using lysate from wild type (WT) mice. Cortex lysate was used as control as expression of Rich2 has been reported in cortex previously. The expression levels of Rich2 in amygdala were slightly higher compared to cortex. The primers used for amplification of Rich2 were placed in a region deleted in the genome of RICH2 Knockout (KO) mice. Absence of Rich2 expression in amygdala of RICH2 KO mice was confirmed. (B)In situ hybridization confirms Rich2 expression in the amygdala in adult WT animals. (C) RICH2 protein was not detected in RICH2 KO mice, but in amygdala of WT mice, & hippocampus & cortex as reported previously. ARP1 is a protein enriched in amygdala & was used as control to ensure proper preparation of amygdala tissue. (D) Immunocytochemistry performed on brain sections of WT mice reveals RICH2 signals co-localizing with HOMER1, a marker for excitatory postsynapses, in the amygdala. Top left: overview indicating magnified area in bottom left & right images. Arrows indicate clearly co-localizing signals (scale bar = 20 μm). Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/article/10.3389/fnmol.2017.00180/full), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - The absence of primary cilia in Kif3a null MEFs has a small effect on hyperosmotic induction of TonEBP without influencing its TAD activity (a) TonEBP/Nfat5 gene expression in wild-type & Kif3a null (Kif3a−/−) MEFs under hyper- (600 mOsm/kg H2O) & hypoosmotic (200 mOsm/kg H2O) conditions. (b,c) Western blot image & corresponding densitometry analyses showing TonEBP levels under different osmotic conditions in wild-type & Kif3a null MEFs. Kif3a null MEFs show slightly attenuated hyperosmotic increase but unaffected hypoosmotic decrease in TonEBP expression. (d,e) TonEBP-TAD (Ton-TAD) activity in wild-type & Kif3a null MEFs under hyperosmotic (d) & hypoosmotic (e) conditions (n = 4). Similar to wild-type cells, Kif3a null MEFs show increase & decrease in TonEBP-TAD activity under hyperosmotic & hypoosmotic conditions, respectively (n ≥ 3 with 3 technical replicates per biological replicate). Data are represented as scatter plots (mean ± SEM). ns = not significant. One-way ANOVA or Kruskal-Wallis test with Sidak’s or Dunn’s multiple comparison test was used based on the distribution of the data to determine statistical significance. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Figs S1 & 3 for un-cropped Western blot images. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664118), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Inhibition of AKT/GSK3 beta / beta -catenin signaling pathway decreased HMGB1-induced EMT.(a) BEAS-2B cells treated w/ 10 μM PI3K inhibitor (LY294002) & GSK-3 beta inhibitor (SB415286) for 10 min, & then treated w/ HMGB1 (300 ng/mL) for 24 h. Protein expression detected by WB analysis. Quantification of E-cadherin, ZO-1, & vimentin performed using ImageJ software. Data are expressed as mean ± SD (n = 5). #p < 0.05, as compared w/ control group. *p < 0.05, as compared w/ HMGB1-treated group. (b) BEAS-2B cells transduced w/ lentiviral-expressed beta -catenin shRNA (1 MOI) for 72 h & selected by puromycin. The mRNA from stable clones expressing beta -catenin-targeting shRNAs analyzed by quantitative real-time PCR. Data are expressed as mean ± SD (n = 5). ##p < 0.01, ###p < 0.001, as compared w/ control group. **p < 0.01, ***p < 0.001, as compared w/ vector group. (c) beta -catenin protein expression detected by WB analysis in a stable beta -catenin shRNA BEAS-2B cell clone. Quantification of protein expression performed using ImageJ software. Data are expressed as mean ± SD (n = 3). ##p < 0.01, ###p < 0.001, as compared w/ control group. **p < 0.01, ***p < 0.001, as compared w/ vector group. (d) A stable beta -catenin shRNA BEAS-2B cell clone treated w/ HMGB1 (300 ng/mL) for 24 h, & protein expression of E-cadherin, ZO-1, & vimentin detected by WB analysis. Quantification of protein expression performed using ImageJ software. Data are expressed as mean ± SD (n = 4). #p < 0.05, as compared w/ control group. *p < 0.05, as compared w/ HMGB1 group. In immunoblotting assay, gels have been run under the same experimental conditions. Then cropped blots incubated w/ different primary antibodies for analysis of signaling pathway. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26739898), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Ang II counteracts IL-6 or TNF alpha inhibition of EC differentiation: the combined effects of Ang II, IL-6, & TNF alpha on VEGF-A-stimulated differentiation of MSCs into ECs were examined. TNF alpha R, IL-6R, AT2R, & VEGFR-2 mRNA expression was analyzed by RT-PCR (n = 3) (a). Sox18 protein levels were measured by Western blot analysis (n = 3) (b). Expression of EC markers was determined by FACS analysis, & a representative grid is shown (n = 3-4) ((c)–(h)). Endothelial tube formation was examined using an angiogenesis assay (n = 3) ((d)–(i)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗p < 0.05 versus naïve MSCs, #p < 0.05 MSCs treated with VEGF-A versus VEGF-A plus Ang II cotreatment, & alpha p < 0.05 versus MSCs treated with VEGF-A & Ang II versus VEGF-A & Ang II plus IL-6 and/or TNF alpha . Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26106428), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - HMGB1-activated AKT/GSK3 beta / beta -catenin signaling pathways.(a) BEAS-2B cells were treated with different doses of HMGB1 for 10 min & expression of different proteins was detected by western blot. (b) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods & analyzed for expression of different proteins by western blot. (c) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods & analyzed for beta -catenin expression by western blot. (d) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods & analyzed for beta -catenin mRNA expression by real-time quantitative PCR. (e) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods & analyzed for nuclear & cytosolic beta -catenin expression by western blot. (f) BEAS-2B cells were treated with 10 μM PI3K inhibitor (LY294002) & GSK-3 beta inhibitor (SB415286) for 10 min, & then treated with HMGB1 (300 ng/mL) for 24 h. beta -catenin expression was assessed by western blot analysis. Quantification of protein expression was performed using ImageJ software. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, as compared with the control group. #p < 0.05, as compared the HMGB1-treatment group. (A,B) (n = 4); (C–F) (n = 3). (g) Immunofluorescence staining of beta -catenin (green) was detected by fluorescence microscopy. Data are expressed as mean ± SD (n = 4). *p < 0.05, as compared with the 0-min group. In immunoblotting assay, gels have been run under the same experimental conditions. Then cropped blots were incubated with different primary antibodies for analysis of signaling pathway. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26739898), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Silencing of endogenous VEGF-A lacks an effect on mesenchymal stem cell differentiation into endothelial cells. Differentiating mesenchymal stem cells (MSCs) were transfected with small interfering RNA (siRNA) directed against vascular endothelial growth factor (VEGF-A) & then examined for endothelial cell (EC) marker expression. Treatment of MSCs with the cocktail of VEGF-A (2 ng/ml) plus angiotensin II (Ang II; 2 ng/ml) for 24 hours induced a synergistic increase in VEGF transcript as compared with VEGF-A alone or Ang II alone (A). VEGF-A mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (A). Standardization experiments demonstrated that 50 nM siRNA directed against VEGF completely abrogated VEGF protein expression (B). Fluorescence-activated cell sorting analysis revealed that there was no significant difference in EC marker expression between differentiating MSCs that received VEGF-A siRNA or scrambled control siRNA (C, D, E). Note that human umbilical vein endothelial cells (HUVECs) were excluded from the statistical analyses. *P <0.05 vs. naïve MSCs. #P <0.05 vs. VEGF-A (2 ng/ml)-treated MSCs or Ang II (2 ng/ml)-treated MSCs alone, n = 3. Image collected & cropped by CiteAb from the following publication (//stemcellres.biomedcentral.com/articles/10.1186/scrt538), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - N-EV & H-EV treatment promote macrophage M2 polarization by delivering miR-21-5p that targets PTEN. a, western blot analysis of PTEN protein expression level in induced macrophages. H/i-miR-EV, monocytes were induced with the presence of EV secreted by miR-21-5p-inhibited, hypoxia pre-challenged MSCs; H-EV + i-miR, monocytes were transfected with miR-21-5p inhibitor-expressing vector before induction with the presence of H-EV. Macrophages induced without MSC-EV were used as negative control (NC). b, c, flow cytometry determining the percentage of CD163+CD206+ cells among total CD68+ cells after induction. N-EV + O/E PTEN or H-EV + O/E PTEN, monocytes were transfected with PTEN overexpressing vector before N-EV or H-EV treatment, respectively. d–f, western blot detecting Akt & STAT3 protein expression as well as their activating phosphorylation (p-Ser473 for Akt & p-tyr705 for STAT3) in macrophages after induction. g–i, ELISA evaluating IL-10, TGF-beta & VEGF-alpha in macrophage culture medium after induction. Macrophages induced with the presence of N-EV were used as negative control in b–i. Tukey’s test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30736829), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Demonstration of the advantages of crude fractionation over whole cell lysis & scale up of the approach. (A) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well plate & subsequently lysed directly in NP40 or RIPA lysis buffer or processed according to the protocol in Figure 2 (Sequential). An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue or by Western blotting & probing with antibodies to various marker proteins of specific intracellular organelles as previously. (B) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish & then transfected the next day in triplicate with 400 ng of a construct encoding V5/6×-HIS tagged ERGIC-53. Cells were extracted 48 hours later either directly in RIPA buffer, directly in NP40 buffer or according to our extraction protocol. Proteins were then partially purified from each extract using Nickel resin (SIGMA #P6611) & eluted proteins were analyzed by 10% SDS PAGE followed by staining with Coomassie blue or by Western blotting & probing with the anti V5 antibody. (C) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish & then transfected the next day in with 1600 ng of pcDNA6/V5-His (Invitrogen) or constructs encoding WT or MUT COMP. Cells were harvested 48 hours later & extracted according to our protocol. (D) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish or 8.4 × 105 cells per well of a 35 mm dish & processed according to the protocol in Figure 2. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting & probing with antibodies to GAPDH, BiP & Lamin A. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20003239), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Notch1, ApoER2 & Dab1 colocalize in hippocampal neurons. (A) Representative image of fluorescent immunolabeling shows that Notch1 is localized in a subset of CA3 neurons, co-expressing Dab1 & ApoER2 (magnified insert). ApoER2 is highly expressed in all CA3 neurons. (B) Fluorescent immunolabeling shows the distribution of Notch1 & Dab1 as compared to Arc/Arg3.1 in CA3 neurons. (B′) Close up of a dendrite showing colocalization of the three proteins (white arrowheads). (C) Western blot analysis on immunoprecipitated samples from whole hippocampal lysate shows that Notch1 displays a strong interaction with Dab1 whereas Notch1 & ApoER2 interaction appears weaker in the preparation (n = 4 independent experiments). (D) Immunolabeling on immunoprecipitated samples from whole cortical lysates show that Notch1 & Dab1 interact. ApoER2 is bound to both Notch1 & Dab1 (n = 3 independent experiments). (E) Co-IP from synaptosomal membrane fractions reveals a stronger interaction between Notch1 & ApoER2, which is reduced in the N1cKO (n = 3 independent pulldowns). In (C–E) GAPDH is used as a loading control for the inputs & to detect contamination in the IP samples. Scale bar in (A) is 50 μm & (B) 25 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26635527), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Reduced expression of UNC5A in FancC−/− cells. a Reduced expression of Unc5A in brain cortex of FancC−/− mice compared to wild-type littermates. Western blotting was performed with the indicated antibodies. Each lane represents a different animal (WT: n = 6; FancC−/−: n = 8). b Bar graph represents ratio of Unc5A normalized to tubulin from WT (n = 6) & FancC−/− mice (n = 8). c Bar graph representation of UNC5A expression normalized to Sdha & Tbp from wild-type (WT) & FancC−/−-derived fibroblasts (n = 3). RLU: Relative light units. d–f HEK293T cells were transfected with UNC5AICD, FANCE & FANCC constructs expressing full-length FANCC (in d), FANCC1–306 (in e) or FANCC307–558 (in f). Constructs were transfected at a molecular ratio of 1:1:1 or with 10 times the molar amount of FANCE, FANCC or UNC5AICD as indicated in the figure. The total amount of transfected plasmid was equalized for all strategies with control empty vectors. Representative immunoblots performed with anti-HA (UNC5AICD), anti-FANCC, anti-FANCE or anti-GAPDH antibodies are shown. Arrows indicate appropriate protein bands. Bar graphs represent the mean fold change ± SEM of UNC5A protein levels normalized to GAPDH compared to 1:1:1 transfection controls in at least 4 separate experiments. *p < 0.05; **p < 0.005 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30213274), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Analysis of GFAP in mouse models of AxD by immunoblotting.Urea-soluble fractions extracted from wild type (GFAP+/+), R236H mutant knockin (GFAPR236H/+) & human wild type GFAP transgenic (GFAPTg) mice (n = 3 for each genotype) were analyzed by immunoblotting with indicated anti-GFAP antibodies (A-D). Immunoblots were also probed with antibodies specific to alpha B-crystallin (F) & GAPDH (E), which was used as a loading control. Representative images were shown, with the antibody used for immunoblotting was indicated above each panel. Approximate molecular weight markers (in kDa) were shown on the right. Dashed lines indicated lanes that samples run on the same gel but were noncontiguous (D & F). Notice that although all anti-GFAP antibodies tested recognized full-length GFAP (A-D), both 2.2B10 (A) & SMI-21 (B) antibodies also detected GFAP degradation products sized between 25 & 35 kDa in GFAPTg mice. The level of alpha B-crystallin (F) was increased in both GFAPR236H/+ & GFAPTg mice compared to wild type controls (GFAP+/+). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0180694), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Knockdown of Ift88 or Kif3a in NP cells does not affect hyperosmotic upregulation of TonEBP. (a) TonEBP/Nfat5 mRNA levels in NP cells with Ift88 knockdown (n ≥ 5). (b) Western blot image showing increased TonEBP expression in response to hyperosmolarity (550 mOsm/kg H2O) independently of Ift88 knockdown. (c) Densitometry analyses of TonEBP with Ift88 knockdown (n ≥ 4). (d) TonEBP/Nfat5 mRNA levels in NP cells with Kif3a knockdown (n ≥ 3). (e) Western blot image showing that hyperosmotic induction of TonEBP is maintained after Kif3a knockdown. (f) Densitometry analyses of TonEBP after Kif3a knockdown (n ≥ 4). Data are represented as scatter plots (mean ± SEM). ns = not significant. One-way ANOVA or Kruskal-Wallis test with Sidak’s or Dunn’s multiple comparison test was used based on the distribution of the data to determine statistical significance. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Fig. S1-1 for un-cropped Western blot images. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664118), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - S166 phosphorylation facilitates necrosome formation.a BMDMs from mice of the indicated genotypes were treated with TNF (T; 10 ng ml−1), SMAC mimetic (S (Birinapant); 1 µM) & Z-VAD-FMK (Z) for 0, 2 & 4 h. RIPK1 immunoprecipitates & total cell lysates (input) were analyzed by immunoblotting with the indicated antibodies. b BMDMs from mice of the indicated genotypes were treated with TNF (T; 10 ng ml−1), SMAC mimetic (S (Birinapant); 1 µM) & Z-VAD-FMK (Z) for 2 & 4 h. Lysates were processed under non-reducing conditions & analyzed for presence of MLKL by immunoblot. c BMDMs from mice of the indicated genotypes were treated with LPS (L; 100 ng ml−1) & Z- VAD-FMK (Z) for 13 & 15 h. Lysates were processed under non-reducing or reducing conditions & analyzed by immunoblot using the indicated antibodies. d BMDMs from mice of the indicated genotypes were treated with Poly(I:C) (P; 0.5 μg ml−1) & Z-VAD-FMK (Z) for 13 & 15 h. Lysates were processed under non-reducing or reducing conditions & analyzed by immunoblot using the indicated antibodies. a, c Representative data of three independent experiments is shown. b, d Representative data of two independent experiments is shown. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32269263), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Notch1 affects CREB phosphorylation & transcription. (A) Immunoblot on whole hippocampal lysates shows a substantial reduction in CamKII in hippocampi of N1cKO as compared to WT. beta -actin is used as a loading control (n = 3 independent experiments). (B) Quantification of CamKII protein expression between N1cKO & WT normalized to beta -actin shows a reduction by 78% in N1cKO hippocampi (n = 6 animals per genotype; Student’s t-test, p < 0.05). (C) Western blot on hippocampal lysates shows ERK1/2 phosphorylation (pERK1/2) in N1cKO is reduced as compared to WT hippocampi. beta -actin is used as a loading control (n = 3 independent experiments). (D) Quantification of ERK1/2 phosphorylation in N1cKO as compared to WT indicates a 76% decrease in KO samples (n = 6 mice per genotype; Student’s t-test, p < 0.05). (E) Immunoblot on whole hippocampal lysates shows a reduction in CREB phosphorylation in N1cKO as compared to WT (n = 3 independent experiments). (F) Bar graph shows a near to significance 56% reduction in CREB phosphorylation in N1cKO hippocampi as compared to WT (n = 4 animals per genotype; Student’s t-test, p = 0.069). (G) Graph summarizing transcripts expression of CREB targets, Egr1, Bdnf & c-fos, in hippocampi from cage control (CC) & environmental exposed (EE) WT & N1cKO mice (n = 3–4 mice per genotype; Student’s t-test, p < 0.05; Black bars compare WT CC w/ WT EE; gray bars compare WT EE w/ N1cKO EE). (H) Model of proposed Notch1 non-canonical signaling regulations of Reelin, NMDA & CREB pathways described in this study. Elements putatively connecting Notch1/Reelin signaling to Notch1/Glutamaterigic signaling are dotted & are based on existing literature. Data are averages ± SEM & *=p < 0.05; 0 signifies p = 0.5. Image collected & cropped by CiteAb from following publication (//pubmed.ncbi.nlm.nih.gov/26635527), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Altered Rho GTPase signaling in amygdala of RICH2 KO mice. (A) Comparison between the GTPase activating protein (GAP) activity of the P2 lysates from WT & RICH2 KO mice. The GAP activity is measured in nmoles/min/mg of small G-protein. First a standard curve is made using the absorbance of KH2PO4 at different concentrations, later the absorbance of WT & RICH2 KO lysates are plotted on the graph. The GAP activity is higher for WT lysates compared to RICH2 KO lysates. (B) In the presence of the small G protein RhoA, the absorbance of WT lysates is higher than that of RICH2 KO lysate, which indicates that RICH2 is acting as a GAP for RhoA in amygdala. (C) There is no difference between the absorbance of WT & RICH2 KO lysates in the presence of RAC1 & CDC42 (D,E) Like RAC1 & CDC42, RAS p21 is not a target G protein for RICH2 in amygdala as there is no difference in the absorbance of WT & RICH2 KO lysates in the presence of RAS p21. (F) Western blot analysis of amygdala P2 lysates reveals that expression of RhoA is significantly higher in RICH2 KO lysates, in comparison with WT lysates (Unpaired t-test, p = 0.0508, n = 3). (G,H) Western blot analysis revealed no alteration in the expressions of Rac1 (G) & Cdc42 (H) between WT & RICH2 KO using amygdala P2 lysates. Image collected & cropped by CiteAb from the following publication (//journal.frontiersin.org/article/10.3389/fnmol.2017.00180/full), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Application of the approach to other cell lines. (A) HT1080 cells (2 × 105) & HeLa cells (1 × 105) were plated in the wells of a 12 well plate & the digitonin concentration required for optimal extraction of the cytosol was determined as in Figure1. (B) The remainder of the protocol from Figure 2 was then applied using 100 μg/ml digitonin as the optimal cytosolic extract in each case. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting & probing with antibodies to GAPDH, BiP & Histone H3 (Used as an alternative nuclear marker due to weakness of the Lamin A signal in HeLa cells). (C) Samples were analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue & then silver staining showing the difference in protein composition of each sample. The bracket alongside the gels marks the position of Histones in the RIPA nuclear extract & the asterisk next to the silver stained gels denotes protein bands unique to the E-RIPA extracts. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20003239), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Optimization of digitonin concentration for the extraction of cytosolic proteins. (A) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well plate & were harvested 48 hours later. Cells were then lysed in 400 μl of buffer containing digitonin at the concentration indicated. Following centrifugation & collection of the supernatant the remaining cell pellet was further extracted in NP40 lysis buffer. An aliquot of each extract was then analysed by 4-12% SDS PAGE (Invitrogen #NP0322) followed by staining with Coomassie blue. (B) The concentration of protein in each extract was determined by colorimetric protein assay (Pierce #23232) & the results were plotted to demonstrate that total protein extracted was the same regardless of the starting concentration of digitonin. (C) Extracted proteins from each fraction were analyzed by Western blot using an anti GAPDH antibody (NOVUS Biologicals #300-221B) as a marker of the cytosol & an anti BiP antibody (SIGMA # G918) as a marker of the endoplasmic reticulum. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20003239), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - CCL2 promotes prostate cancer cell migration & induces CCL22 secretion, which is a ligand of CCR4A. Prostate cancer cells are placed in transwell inserts & treated with CCL2 (0–30 ng/ml). After 24-h incubation, PC-3 & DU145 cells that had migrated through the membrane are stained. The mean OD value is read using a microreader at 595 nm. Migration of LNCaP cells is assessed with a wound-healing assay. Data are presented as mean ± SD. B. DU145 cells are co-cultured with THP-1 & U937 cells & treated with CCL2 (30 ng/ml) for 24 h, CM is collected, & CCL17 & CCL22 levels are analyzed using ELISA. The mean OD value is read using a microreader at 450 nm, & data are presented as mean ± SD. C, D. Total RNA & protein are extracted from prostate cancer cells, & CCR2 & CCR4 gene & protein expression levels are analyzed using PCR (C) & western blot (D). E. Prostate cancer cells (1.0 × 105 cells/well) are seeded into 6-well plates & cultured until they reach 60%–70% confluence. Cells are incubated with anti-CCR2 or anti-CCR4 antibody & detected using a second antibody conjugated with FITC (green). Cells are counterstained with 4',6-diamidino-2-phenylindole (blue). Adjustments of brightness, contrast, & size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. All experiments are performed in triplicate, & mean values are shown. *p < 0.05, **p < 0.01, ***p < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28039457), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Stable knockdown of Ift88 or Kif3a inhibits formation of NP cell primary cilia. (a) Ift88 mRNA levels in NP cells transduced with control (ShCtr) or two different ShIft88 clones were measured by qRT-PCR to confirm the knockdown (n ≥ 5). (b) Western blot image showing significant reduction of IFT88 protein levels after the knockdown of Ift88. (c) Densitometry analyses of Western blots confirm significant knockdown of IFT88 (n ≥ 5). (d–f) qRT-PCR, Western blot, & corresponding densitometry analyses, show significant downregulation of KIF3A after stable knockdown using two different ShKif3a clones (n ≥ 4). (g) Acetylated alpha -tubulin immunofluorescence staining after lentiviral transduction of ShIft88 or ShKif3a shows inhibition of primary cilia formation in majority of rat NP cells. Scale bar = 75 μm. White arrowheads point to primary cilia. (h,i) Quantitation of percentage of NP cells with primary cilia & primary cilium length after stable silencing of Ift88 or Kif3a (n = 3; at least 150 cells/group). Data are represented as scatter plots (mean ± SEM). ns = not significant. One-way ANOVA or Kruskal-Wallis test with Sidak’s, Holm-Sidak’s, or Dunn’s multiple comparison test was used based on the distribution of the data to determine statistical significance. For statistical comparison of the percentages of NP cells with primary cilia, Fisher’s exact test was used. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Fig. S1–1 for un-cropped Western blot images. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31664118), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - CCL2 increases not only CCL22 but also CCR2 & CCR4A. Prostate cancer cells are treated with CCL2 (30 ng/ml) for 24 h, & western blot for CCR2 is performed. B. Prostate cancer cells are treated with CCL2, CCL17, & CCL22 (30 ng/ml) for 24 h, & western blot for CCR4 is performed. C. Prostate cancer cells are co-cultured with U937 or U937-M cells for 24 h, & western blot for CCR4 is performed. D. Prostate cancer cells are placed in transwell inserts & treated with CCL17 & CCL22 (0–30 ng/ml). After 24-h incubation, PC-3 & DU145 cells that had migrated through the membrane are stained. The mean optical density (OD) value is read using a microreader at 595 nm. Migration of LNCaP cells is assessed with a wound-healing assay. Adjustments of brightness, contrast, & size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. Data are presented as mean ± SD. All experiments are performed in triplicate, & mean values are shown. *p < 0.05, **p < 0.01, ***p < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28039457), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - ATR2 siRNA transfection & immunophenotyping for EC markers. I Concentration selection for siRNA transfection. Three different concentrations (10, 35, & 50 nM) of ATR2 siRNA were used according to the manufacturer’s protocol. Western blot analysis showed inhibition of ATR2 by 10, 35, & 50 nM of ATR2 siRNA. However, 50 nM of ATR2 siRNA showed the highest inhibition among all three different concentrations (A). ATR2 silencing by siRNA transfection with EGM compared with AMSCs with EGM & EGM + scrambled siRNA (negative control) (B). GAPDH was used as a housekeeping gene. II Flow cytometric analysis of PECAM1 (CD31) in four different groups; control group with EGM (A), AMSCs with EGM & MMP-2 siRNA (B), AMSCs with EGM & MMP-14 siRNA (C), & HUVECs as the positive control (D). Cell transfection with 5 μM of ATR2 siRNA for EGM (E), AMSCs with EGM & MMP-2 siRNA (F), & AMSCs with EGM & MMP-14 siRNA (G). Flow cytometry data were analyzed to show the significant differences between the groups (H). III Flow cytometric analysis of VE-cadherin (CD144) in four different groups: control group AMSCs with EGM (A), AMSCs with EGM & MMP-2 siRNA (B), AMSCs with EGM & MMP-14 siRNA (C), & HUVECs as the positive control (D). Cell transfection with 5 μM of ATR2 siRNA for EGM (E), AMSCs with EGM & MMP-2 siRNA (F), & AMSCs with EGM & MMP-14 siRNA (G). Flow cytometry data were analyzed to show the significant differences between the groups (H). **p < 0.01. EBM endothelial cell basal medium, EGM endothelial cell growth medium, MMP matrix metalloproteinase, ATR2 angiotensin receptor R2, HUVEC human umbilical vein endothelial cell Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28499402), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Protein expression of CTGF, TIMP‐1, TGF‐ beta 1, alpha ‐SMA & collagen type 1 in WT & MMP‐13 KO mice treated without & with NDMA. (A) Western blotting for the proteins of full‐length CTGF & its C‐terminal fragments, TIMP‐1, active TGF‐ beta 1, alpha ‐SMA & collagen type 1 in the liver tissues from WT & MMP‐13 KO mice. The images are representative of six Western blots per each group. (B) Quantitative analysis of Western blot images. The data are mean ± S.D. of six images per group. ***P < 0.001 NDMA‐treated WT mice versus control untreated WT mice & NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; **P < 0.01 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; *P < 0.05 NDMA‐treated MMP‐13 KO mice versus untreated MMP‐13 KO mice; #P < 0.001 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice; @, P < 0.05 NDMA‐treated MMP‐13 KO mice versus NDMA‐treated WT mice. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28782260), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Endothelial responses provoked by Hb-lipid interactions are attenuated by sulfide donors. (a) OxLDL (200 μg/mL) was incubated with sulfide donors NaSH, GYY4137, AP67, & AP72 at the concentrations of 20 & 200 μmol/L at 37°C for 24 hours. Confluence HAoECs were exposed to the LDL samples for 4 hours then cell viability was determined by MTT assay. Representative experiment, n = 3, each performed in 8 wells in parallel. (b) OxLDL (200 μg/mL) was incubated with sulfide donors NaSH, GYY4137, AP67, & AP72 at the concentration of 20 & 200 μmol/L at 37°C for 24 hours. Confluence HAoECs were exposed to LDL samples (50 μg/mL) for 8 hours. HO-1 & HO-2 protein expressions were determined by Western blotting. Representative experiment, n = 3. (c–e) Hb (10 μmol/L) was incubated with H2O2 (50 μmol/L) in the presence or absence of sulfide donors NaSH, GYY4137, AP67, & AP72 (200 μmol/L) at 37°C for 90 minutes. (c) Confluence HAoECs were exposed to the obtained Hb samples for 8 hours & VCAM-1 expression was determined by Western blotting. Representative experiment, n = 3. (d, lower panel) Confluence HAoECs cultured in ECIS plates were exposed to the obtained Hb samples & transendothelial electrical resistance was monitored by ECIS instrument for 3 hours. Representative experiment, n = 3, each performed in triplicate (d, upper panel). HAoECs grown on coverslips, upon reaching confluence cells were exposed to the Hb samples for 8 hours. Cells were stained for F-actin (phalloidin-TRITC, red) & DNA (Hoechst, blue). White arrows show intercellular gaps. Representative image, n = 5. ∗∗ indicates statistical significance (p < 0.001). ∗∗∗ indicates statistical significance (p < 0.0001). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29560080), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Upregulation & accumulation of the endogenous mouse GFAP in the GFAPTg mice.Urea-soluble fractions prepared from whole brains of GFAP+/+ wild type (A, lanes 1–3) & GFAPTg (A, lanes 4–6) mice were analyzed by immunoblotting using the anti-mouse GFAP antibody. Immunoblots probed with anti-GAPDH antibody was used as a loading control. Note that the endogenous mouse GFAP was significantly increased in the GFAPTg mice (A, lanes 4–6) compared to wild type controls (A, lanes 1–3). Approximate molecular weight markers (in kDa) were shown on the left. The distributions of human GFAP in relation to the endogenous mouse GFAP in brain sections of wild type (GFAP+/+, B) & GFAPTg (C-H) mice were visualized by double-label immunofluorescence microscopy using anti-mouse GFAP & SMI-21 antibodies. The immunofluorescence for human GFAP was in the green channel (C & F), whereas the counterstaining for mouse GFAP was in the red channel (D & G). Merged images showed the superimposition of both the green & red signals, with overlapping area appearing yellow (B, E, & H). Nuclei were revealed by staining with DAPI (B, E, & H). In wild type mice, normal appearance of GFAP in astrocytes was readily stained with the anti-mouse GFAP antibody (B), with very little staining with the SMI-21 antibody. Conversely, GFAPTg mice showed numerous Rosenthal fiber-like GFAP aggregates in the granular cell layer of the cerebellum (C-E) & the pial surface of the cortex (F-H), which were immunopositive for both mouse & human GFAP. Bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0180694), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - IL-6 negatively regulates EC differentiation: the effect of IL-6 on VEGF-A-stimulated differentiation of MSCs into ECs was examined. IL-6R & VEGFR-2 mRNA expression was analyzed by RT-PCR & normalized to GAPDH (n = 3) (a). Sox18 protein levels were measured by Western blot analysis & normalized to GAPDH (n = 3) (b). Expression of EC markers was determined by FACS analysis, & a representative grid is shown (n = 3–6) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay (n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗p < 0.05 versus naïve MSCs & #p < 0.05 MSCs treated with VEGF-A versus VEGF-A plus IL-6 cotreatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26106428), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Demonstration of the advantages of crude fractionation over whole cell lysis & scale up of the approach. (A) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well plate & subsequently lysed directly in NP40 or RIPA lysis buffer or processed according to the protocol in Figure 2 (Sequential). An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue or by Western blotting & probing with antibodies to various marker proteins of specific intracellular organelles as previously. (B) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish & then transfected the next day in triplicate with 400 ng of a construct encoding V5/6×-HIS tagged ERGIC-53. Cells were extracted 48 hours later either directly in RIPA buffer, directly in NP40 buffer or according to our extraction protocol. Proteins were then partially purified from each extract using Nickel resin (SIGMA #P6611) & eluted proteins were analyzed by 10% SDS PAGE followed by staining with Coomassie blue or by Western blotting & probing with the anti V5 antibody. (C) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish & then transfected the next day in with 1600 ng of pcDNA6/V5-His (Invitrogen) or constructs encoding WT or MUT COMP. Cells were harvested 48 hours later & extracted according to our protocol. (D) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish or 8.4 × 105 cells per well of a 35 mm dish & processed according to the protocol in Figure 2. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting & probing with antibodies to GAPDH, BiP & Lamin A. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20003239), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Co-culture of macrophages & prostate cancer cells increases prostate cancer cell migration & invasion & induces CCL2 secretionA. U937 differentiation to M2 macrophages is determined using western blot analysis. CCR7 (an M1-type macrophage marker) & CD206 (an M2-type macrophage marker) are assayed using proteins extracted from U937 cells treated with PMA for 24 h & exposed to CM of prostate cancer cells. B. Prostate cancer cells are placed in transwell inserts, & CM of U937 & U937-M cells is added. C. Prostate cancer cells are placed in transwell inserts with Matrigel®-coated membranes in the upper compartment, & U937-M cells are placed in the lower compartments. After 24-h incubation, the cells that had migrated through the membrane are stained. The mean optical density (OD) value is read using a microreader at 595 nm. Data are presented as mean ± SD. D. Chemokine arrays comparing the CM of PC-3 cells in monoculture & the CM of PC-3 cells co-cultured with monocyte cells. E. Prostate cancer cells are co-cultured with U937 cells for 24 h, CM is collected, & CCL2 levels are analyzed using ELISA. Adjustments of brightness, contrast, & size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. The mean OD value is read using a microreader at 450 nm, & data are presented as mean ± SD. All experiments are performed in triplicate, & the mean values are shown. *p < 0.05, **p < 0.01, ***p < 0.001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28039457), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - HMGB1-induced EMT in human airway epithelial cells.Human primary airway epithelial cells & BEAS-2B cells were treated with different doses of HMGB1 for mRNA analysis at different time points, & at 24 h for protein analysis. E-cadherin, ZO-1, & vimentin mRNA expression was detected by real-time quantitative PCR in (a) human primary airway epithelial cells & (c) in BEAS-2B cells after HMGB1 treatment (300 ng/mL) for 24 h. E-cadherin, ZO-1, & vimentin protein expression in (b) human primary airway epithelial cells andBEAS-2B cells (d) was assessed by western blot analysis. Data are expressed as mean ± SD (n = 3–5). *p < 0.05, **p < 0.01, ***p < 0.001, as compared with the control group. (e) TGF-beta 1 expression in cell lysate (LAP) was assessed by western blot analysis & in cell medium (active form) by ELISA. Quantification of protein expression was performed using ImageJ software. Data are expressed as mean ± S.D. (n = 4). In immunoblotting assay, gels have been run under the same experimental conditions. Then cropped blots were incubated with different primary antibodies for analysis of EMT markers expression. Original blots of (b) are presented in Supplementary Fig. 3. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26739898), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Expression of CSE in human aortic valves. (a) Western blot of CSE protein from AS valve (N = 12) lysates & AI valve (N = 9) lysates (left panel) were performed. Representative CSE protein levels of three AS & three AI valves lysates were shown. Relative CSE levels were assessed by densitometry analyses of band intensities of CSE western blots normalized to GAPDH (panel right). (b) Zn2+ precipitated sulfide content under alkaline conditions was measured in AI (N = 18) & AS (N = 18) valve tissues normalized to protein content of the individual samples. (c) VIC derived from different AI (N = 6) & AS (N = 9) patients were cultured in calcification medium. CSE expression was measured after the initiation of calcification. Results were normalized to GAPDH of the samples. (d) Double immunohistochemistry of CSE‐SMA & CSE‐ALP was shown with two different magnifications (×400 magnification & ×1,000 magnification). Data shown are means ± SEM of N experiments. *P < .05, significantly different as indicated Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31017307), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Demonstration of the advantages of crude fractionation over whole cell lysis & scale up of the approach. (A) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well plate & subsequently lysed directly in NP40 or RIPA lysis buffer or processed according to the protocol in Figure 2 (Sequential). An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue or by Western blotting & probing with antibodies to various marker proteins of specific intracellular organelles as previously. (B) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish & then transfected the next day in triplicate with 400 ng of a construct encoding V5/6×-HIS tagged ERGIC-53. Cells were extracted 48 hours later either directly in RIPA buffer, directly in NP40 buffer or according to our extraction protocol. Proteins were then partially purified from each extract using Nickel resin (SIGMA #P6611) & eluted proteins were analyzed by 10% SDS PAGE followed by staining with Coomassie blue or by Western blotting & probing with the anti V5 antibody. (C) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish & then transfected the next day in with 1600 ng of pcDNA6/V5-His (Invitrogen) or constructs encoding WT or MUT COMP. Cells were harvested 48 hours later & extracted according to our protocol. (D) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish or 8.4 × 105 cells per well of a 35 mm dish & processed according to the protocol in Figure 2. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting & probing with antibodies to GAPDH, BiP & Lamin A. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/20003239), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Effect of forskolin on the surface expression of CFTR. (A) Quantitation & representative blot of cell surface biotinylation of CFTR in HEK‐CFTR cells that were treated with forskolin (FSK; 10 μmol/L) for 1, 4, & 10 min. GAPDH was used as a control to determine if there was any contamination of biotinylated protein in the nonbiotinylated fractions (Ab: monoclonal mouse‐anti‐CFTR‐C & anti‐GAPDH; 1:1000 & 1:2000 dilution, respectively; estimated sizes: for CFTR: 165 kDa & GAPDH: 36 kDa). (B) Effect of FSK on CFTR location in live HEK‐CFTR cells. Cells grown in 35‐mm glass‐bottom dishes were stained as described in the Methods. Images were captured before & after the treatment of FSK as time series. Snapshots at 0, 1, 4, & 10 min are shown. Green: CFTR; red: WGA; & blue: Dapi. White arrows: colocalization of CFTR & WGA; green arrows: CFTR vesicles. n ≥3 for all experiments. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25263207), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - HER2/HER3 & AXL expression & phosphorylation analysis.A) Representative western blots of total cell lysates of HCC827 & HCC4006 parental cell lines (P) & their derived ERL-resistant cell lines. Arrows indicate the expected molecular weight size. Total cell lysates loaded were 40 μg for AXL & pAXL analyses & 25 μg for the others. B) qPCR analysis of AXL mRNA normalized to rp-L31 mRNA & expressed relative to the levels in parental cell lines set as 1 (mean ± SD of triplicate determinations). Western blots & qPCR data are representative of those obtained respectively from 3 & 2 independent analysis. C) Dose-effect curves were calculated using CompuSyn software & plotting the entered Fa values against the entered dose values. For combination treatments, the combined drugs dose was entered. Each data point represents the mean of 3 replicates. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0143333), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Ang II positively regulates VEGF-A-mediated EC differentiation: the effect of Ang II on VEGF-A-stimulated differentiation of MSCs into ECs was examined. AT2R & VEGFR-2 mRNA expression was analyzed by RT-PCR (n = 3) (a). Sox18 protein levels were measured by Western blot analysis (n = 3) (b). Expression of EC markers was determined by FACS analysis, & a representative grid is shown (n = 5-6) ((c)–(g)). Endothelial tube formation was examined using an angiogenesis assay (n = 3) ((d)–(h)). Experiments were performed with samples taken from independent BM-MSC cultures from separate microswine. HUVECs were excluded from statistical analyses. Data are shown as mean ± SD. ∗p < 0.05 versus naïve MSCs & #p < 0.05 MSCs treated with VEGF-A versus VEGF-A plus Ang II cotreatment. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26106428), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Expression of Npr2 & cGKI alpha in DRGs at different developmental stages. (A,B) Western blot of membrane or cytosolic fractions of DRGs using antibodies to Npr2 or to cGKI alpha , respectively. The heavy chain of clathrin or GAPDH served as loading control. (C–E) Sections through thoracic DRGs at different stages. Localization of anti-beta Gal staining indicating Npr2-expression is demonstrated in the Npr2wt/LacZ-mouse reporter & localization of cGKI alpha by a polyclonal antibody to mouse cGKI alpha . Scale bars in (C1,D1), 50 μm; in (C2-C5,D2-D5), 100 μm; in (E) 25 μm. (F) Quantification of Npr2-positive & cGKI alpha -positive neurons in DRGs counterstained with an antibody to PGP9.5 at different stages. (G–I) A small proportion of Npr2-positive DRG neurons at P45 & P75 express trkA, parvalbumin, CGRP or IB4. Scale bars in (H,I), 20 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29472841), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Bub1-KD & Bub1-T589A display increased residency in the cytosol.(a) Mitotic control (siGL2) & Bub3-depleted (siBub3) cells were fixed & stained with anti-H2A-pT120 (red), anti-Sgo1 (green) & anti-CREST (blue). (b) Quantification of the localization of H2A-pT120 & Sgo1 signals. Data represent the mean±s.e. of three independent experiments. Eighty to 300 cells were scored per condition per experiment. (c) Images & (d) quantification (normalized average pixel intensity); low (1–1.2), medium (>1.2 to ≤1.3) & high (>1.3) of 3 × MYC-GFP-Bub1 signal & localization in live cells synchronized in mitosis by a thymidine release. Data represent the mean±s.e. of 3 independent experiments, with 58–61 cells measured per condition. Significance was measured for the high group by one-way analysis of variance (ANOVA) & pairwise t-test (Holm–Sidak). (e) Scatter plot of the cytoplasm versus kinetochore GFP levels of individual cells from each of the stable cell lines. The number of cells, R2 (measure of the goodness-of-fit) & significance (one-way ANOVA) are indicated. (f) Western blottings showing levels of the 3 × MYC-GFP-Bub1 proteins in the stable cell lines in whole cell extracts (left) & in cytoplasmic extracts (right). Scale bar, 10 μM. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms9364), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Notch1, ApoER2 & Dab1 colocalize in hippocampal neurons. (A) Representative image of fluorescent immunolabeling shows that Notch1 is localized in a subset of CA3 neurons, co-expressing Dab1 & ApoER2 (magnified insert). ApoER2 is highly expressed in all CA3 neurons. (B) Fluorescent immunolabeling shows the distribution of Notch1 & Dab1 as compared to Arc/Arg3.1 in CA3 neurons. (B′) Close up of a dendrite showing colocalization of the three proteins (white arrowheads). (C) Western blot analysis on immunoprecipitated samples from whole hippocampal lysate shows that Notch1 displays a strong interaction with Dab1 whereas Notch1 & ApoER2 interaction appears weaker in the preparation (n = 4 independent experiments). (D) Immunolabeling on immunoprecipitated samples from whole cortical lysates show that Notch1 & Dab1 interact. ApoER2 is bound to both Notch1 & Dab1 (n = 3 independent experiments). (E) Co-IP from synaptosomal membrane fractions reveals a stronger interaction between Notch1 & ApoER2, which is reduced in the N1cKO (n = 3 independent pulldowns). In (C–E) GAPDH is used as a loading control for the inputs & to detect contamination in the IP samples. Scale bar in (A) is 50 μm & (B) 25 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26635527), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GAPDH Antibody (1D4) [NB300-221] - Depletion of RAD21 & other cohesin subunits leads to differentiation of ESCs.(A): qPCR analysis of esiRNA knock-down efficiency for indicated genes (48 h RNAi, n = 3, error bars denote s.d. & *, ** indicate p<0.05 & 0.01 respectively). Transfection of a non-targeting esiRNA (Luc) was used as a control. Numbers adjacent to the gene names indicate independent, non-overlapping esiRNAs transfected. (B): Western Blot analysis of esiRNA knock-down efficiency for indicated genes (48 h RNAi; esi-1 & esi-2 indicate independent, non-overlapping esiRNAs transfected). Transfection of a non-targeting esiRNA (Luc) was used as a control. GAPDH served as a protein loading control. (C): Alkaline phosphatase staining of ESCs, which had been transfected with esiRNAs targeting RAD21, SMC1a & SMC3 (72 h post RNAi) showed a strong differentiation phenotype compared to a control (Luc). Nanog depletion served as a positive control for ESC differentiation. Scale bars correspond to 100 µm. (D): Quantification of alkaline phosphatase staining (n = 3) separated into undifferentiated (green), mixed (yellow) & differentiated colonies (red). (E+F): qPCR validation of expression changes of (E) stem cell maintenance genes & (F) lineage marker genes upon knock-down of RAD21 (two independent esiRNAs) & Luc control (48 h RNAi, n = 3, error bars denote s.d. & *, **, *** indicate p<0.05, 0.01 & 0.001 respectively). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/21589869), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Role of the TRIM11-USP14 axis in cell growth & apoptosis. a Levels of endogenous TRIM11 in HCT116 cells grown at 43 °C for 0 or 90 min, or at 43 °C for 90 min, & then at 37 °C for 60 or 120 min. b, c Levels of endogenous TRIM11 (b), or Flag-TRIM11, the indicated proteasome subunits, & USP14 (c) in the SN & P fractions of HCT116 cells with & without heat shock at 43 °C for 90 min. d, g TRIM11 overexpression (d) or knockdown (g) HCT116 cells & the corresponding control cells grown under unstressed, heat shock, & heat shock plus recovery conditions were analyzed for K48 polyUb conjugates & caspase-3 activation. e, f, h Apoptosis in TRIM11 overexpression (e, f) or knockdown (h) HCT116 cells, & the corresponding control HCT116 cells, treated with & without heat shock. i, j Caspase-3 activation (i) & apoptosis (j) in HCT116 cells expressing the indicated proteins and grown under normal or heat stress conditions. k–m K48 polyUb conjugates, caspase-3 activation (k), amyloid-like fibrils (l), & apoptosis (m) in HCT116 cells expressing the indicated proteins and grown under normal & heat stress conditions. In e, f, h, j, l, & m, data represent mean ± SEM (n = 3 unless otherwise indicated). *P < 0.05; **P < 0.01; ***P < 0.001. Uncropped blots are presented in Supplementary Fig. 14 Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-018-03499-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Reactivity:

Hu, Mu, Rt, Po, Av, Bv, Ca, Ch, ChHa, Dr, Eq, Fe, Ha, Ma-Op, Pm, Rb, Sh, Ze

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Reactivity: Hu, Mu, Rt, Po, Av, Bv, Ca, Ch, ChHa, Dr, Eq, Fe, Ha, Ma-Op, Pm, Rb, Sh, Ze

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, , , , , , ,

Applications: , , , , , , ,

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Mouse Monoclonal

Host: Mouse Monoclonal

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Sizes: Sample Size Available

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BSA Free	NB300-221SS

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At	=	A. thaliana	SyHa	=	Golden Syrian Hamster
All	=	All Species	Ha	=	Hamster
An	=	Animal	Hu	=	Human
ArHa	=	Armenian Hamster	I	=	Insect
Av	=	Avian	Ll	=	Llama
Bb	=	Baboon	Ma	=	Mammal
Ba	=	Bacteria	Mk	=	Monkey
Bv	=	Bovine	Mu	=	Mouse
Ca	=	Canine	Multi	=	Multi-species
Ce	=	C. Elegans	NA	=	Non-species specific
Ch	=	Chicken	Or	=	Orangutan
ChHa	=	Chinese Hamster	Pl	=	Plant
Cm	=	Cynomolgus Monkey	Pm	=	Primate
Do	=	Donkey	Po	=	Porcine
Dr	=	Drosophilia	Rb	=	Rabbit
Ec	=	E. Coli	Rt	=	Rat
Eq	=	Equine	RM	=	Rhesus Macaque
Fe	=	Feline	Sh	=	Sheep
Ft	=	Ferret	Vb	=	Vertebrate
Fi	=	Fish	Vi	=	Virus
Fu	=	Fungi	Xp	=	Xenopus
Ge	=	Gerbil	Ye	=	Yeast
GP	=	Guinea Pig	Ze	=	Zebrafish
Gt	=	Goat

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