Product Search for Immunoprecipitation: Novus Biologicals

NB100-2220

LC3B Antibody - BSA Free

(51 Reviews)

(1804 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>Contribution of autophagy to lipid droplet formation in WT and KO NSC34 cells.(A) WT and KO NSC 34 cells were treated with (+) or without (-) bafilomycin A (bafA) under basal or HBSS starvation conditions for 4 h and blotted for p62, LC3, and GAPDH. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32321733) licensed under a CC-BY license.

TMEM16A inhibited AngII-induced autophagy in aortas. (D) Representative immunofluorescence staining of LC3 (green) and a-SMA (red) in aortas. Scale bars, 20 um. Quantification of LC3B puncta. Five random fields (2,500 um2/filed) were measured in one section. **P 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 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.

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] - 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] - 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
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)

(1060 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.

HIF1alpha/PFKFB3 is upregulated in beta-cells of HIP rats and humans with type 2 diabetes. Representative Western blot of PFKFB3 and HIF1alpha levels in nuclear-enriched- and whole cell extracts from non-diabetic (ND) and 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 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 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] - 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.

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

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] - 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] - 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.

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.

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] - 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] - 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] - 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] - 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
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)

(866 Publications)

0.1 ml

NB100-122

WNT11 is induced by hypoxia or hypoxic mimetics in different cell types. Immunoblot analyses of HeLa cells under normal air or hypoxia for 24 hrs. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>HIF-1alpha is the predominant transcriptional regulator of WNT11 expression during hypoxia. EMSCs isolated from the indicated mouse genotypes were infected with lentivirus expressing GFP or Cre recombinase. Non-infected cells and GFP infected cells served as controls. Immunoblot analyses of EMSCs derived from the indicated genotypes treated with 0.1 mM DMOG for 24 hrs. Attenuated WNT11 expression in Hif-1alpha KO EMSCs (lenti-Cre infected Hif-1af/f). Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY license.

<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.

Expression of hypoxia-inducible alpha subunits in normal and diseased lung tissue. HIF-2alpha expression are more evident in fibroblasts from idiopathic pulmonary fibrosis (d) than from lung tissue affected by other inflammatory conditions (i.e. chronic bronchitis, panel e) or normal lung tissue (f); as demonstrated by a higher proportion of positive fibroblasts (open arrows) than negative ones (solid black arrows). Image collected and cropped by CiteAb from the following publication (//respiratory-research.biomedcentral.com/articles/10.1186/s12931-019-1100-4) licensed under a CC-BY license.

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>Immunoblot validation of HIF2A and PHD2 KO clones using HIF2A (#NB100-122; dilution: 1/300), PHD2 (#NB100-137; dilution: 1/500). To blot HIFs factor cells were first pre-treated for 5 h with CoCl2 300 uM before protein extraction, a condition that promotes HIF factor accumulation. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-018-06988-3) licensed under a CC-BY license.

<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.

Induced WNT11 expression with tumor hypoxia and WNT11 regulates tumor growth. Bevacizumab increased expression of HIF-1alpha and HIF-2alpha and WNT11. First 10 lanes are control tumors, and the last 10 lanes are tumors from bevacizumab-treated animals. Lysates from whole tissue and nuclei are indicated. Alpha -Tubulin, actin and lamin A/C are loading controls. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY license.

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 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
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)

(795 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>Effect of RK-33 on radiation-induced DNA damage. A Immunofluorescence images showing 53BP1 and gamma-H2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 uM RK-33, 12 h before radiation. Overlap of 53BP1 and gamma-H2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 um. Image collected and cropped by CiteAb from the following publication (//embomolmed.embopress.org/cgi/doi/10.15252/emmm.201404368) licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Lamin A/C-53BP1 interaction is regulated in a DNA damage-dependent manner. U2OS/GFP-lamin A cells were pretreated with 10 um ATMi f for 1 h before exposure to IR (10 Gy, 1 h recovery). Cell extracts were then subjected to immunoprecipitation using GFP-Trap beads, and bound complexes were then analyzed by immunoblotting using 53BP1 and GFP antibodies. WCE represents 1% input. Image collected and cropped by CiteAb from the following publication (//doi.wiley.com/10.1111/acel.12258), licensed under a CC-BY license.

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>MUS81 inhibition in BRCA2-deficient cells causes accumulation of 53BP1 nuclear bodies and G1 arrest. H1299 cells carrying a DOX-inducible BRCA2 shRNA were transfected with control or MUS81 siRNAs. Representative images of cells processed 72 h later for immunostaining with 53BP1 Antibody (green) and anti-cyclin A (red) antibodies. DNA was counterstained with DAPI. Scale bar, 10 um. Image collected and cropped by CiteAb from the following publication (//www.nature.com/doifinder/10.1038/ncomms15983), licensed under a CC-BY license.

<b>Biological Strategies Validation. </b>Pot1a prevents DDR in HSCs. Telomeric DDR in 8 week-old LSK cells upon Pot1a knockdown. Immunocytochemical staining of TRF1 (green). Foci co-stained with TRF1 and 53BP1 were identified as TIFs. Nuclei were stained with TOTO3 (blue). Scale bar, 2 um. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-017-00935-4), licensed under a CC-BY license.

Lamin A/C-53BP1 interaction is regulated in a DNA damage-dependent manner. U2OS cells were transfected with siCTRL or siLMNA and subjected to laser micro-irradiation, fixed 15 min later and then processed for immunofluorescence with gamma-H2AX and 53BP1 antibodies. Scale bar, 10 um. Image collected and cropped by CiteAb from the following publication (//doi.wiley.com/10.1111/acel.12258), licensed under a CC-BY license.

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.

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 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 0.05. 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 0.05. (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 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.

$Immunocytochemistry/ Immunofluorescence: 53BP1 Antibody - BSA Free [NB100-304] - Loss of functional PRPF31 induce RNA:DNA hybrid dependent genomic instability but not in mice retinal neurons.(a andb) gamma H2AX & 53BP1 foci analysis in PRPF31 siRNA-transfected RPE-1 cells. (c & d) gamma H2AX & 53BP1 foci analysis in vasculo-stromal fraction derived primary cells fromPrpf31+/A216P mice (Prpf31-ki). (e) gamma H2AX & 53BP1 foci analysis in retina fromPrpf31+/A216P mice on postnatal day 20. All column bars represent the mean. For (a-d) “n”, mentioned on respective column, signify number of cells analyzed from two independent experiments. For (e) n=16 for each column & signify number of retinal sections analyzed; acquired from n=4 eyes. Error bars represent Standard error of Mean (SEM). *P≤0.05; **P 0.9999, ****P 0.05). ns = not significant. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.21145), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: 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] - 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 1.0 mm, >2.0 mm) are shown. Data are expressed as the mean ± SD (n = 3, *p 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 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] - 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, Bt, Bv, Ca, Fi, Gt, Pm, Po, Pm, Fe, Pm-Or, Rb, Sh

Species Glossary

Reactivity: Hu, Mu, Rt, Bt, Bv, Ca, Fi, Gt, Pm, 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
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)

(615 Publications)

0.1 ml

NB600-501

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>Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - MMP-11 expression in oral cancer cell lines and the MMP-11 knockdown in SCC9 cells reduce cell migration. Western blot analysis showing the expression of MMP-11 protein in five oral cancer cell lines. Beta-Actin is included as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0113129), licensed under a CC-BY license.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - HSP90 and HSP70 interact with PABPN1. Interaction of PABPN1 with HSP90. Lysates from HEK293 cells transfected with Flag-HSP90 and GFP-tagged A17-PABPN1 were immunoprecipitated with an anti-Flag antibody. Immunoprecipitates (IP) were analyzed by Western blot (IB).Beta-Actin is included as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0138936), licensed under a CC-BY license.

Western Blot: beta-Actin Antibody (AC-15) [NB600-501] - GFAP and IL-6 are elevated in cultures of spinal cord compared to cortical astrocytes. ((G, H) Analysis of protein homogenates from brain or spinal cord astrocyte cultures also demonstrated higher levels of GFAP expression, but no differences in ALDH1L1. Beta-Actin is included as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0180697), licensed under a CC-BY license.

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). 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 0.1, 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 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, One‐way ANOVA). ZO‐1 level: P 0.05, One‐way ANOVA). ZO‐1 level: P 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] - 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] - 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] - 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] - 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] - 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] - 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] - 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] - 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] - 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] - 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: 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.

Reactivity:

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

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Bv, Ca, Ch, Fe, Fi, Gp, 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
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 549	NB110-67828JF549
Janelia Fluor 646	NB110-67828JF646
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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NB100-479

HIF-1 alpha Antibody - BSA Free

(28 Reviews)

(538 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.

<b>Biological Strategies Validation. </b>Normoxic pre-treatment sensitizes glioma cells to radiation after graded chronic hypoxic (GCH) exposure. The results of anchorage-independent colony forming assays are shown for U87, U87-luc, GL261 glioma cells and 0308 GSCs after 5 Gy radiation exposure under varying O2 conditions. For easier comparisons among cell types, raw values are expressed as a % of the cell type's negative (non-irradiated) control and the means & SEMs are plotted. Each result represents at least 3 independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p 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; *p 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.

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.

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] - 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] - 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
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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NB600-308

GFP Antibody - BSA Free

(18 Reviews)

(307 Publications)

0.1 mg

NB600-308

Analysis of GFP in transgenic mouse pancreas in OCT. The blue region in the center is an islet which does not express GFP and is negative. Image from verified customer review.

Analysis of GFP tagged PKA subunits over-expressed in HEK293T cells using GFP antibody. Image from verified customer review.

Staining of GFP+ hepatocytes (brown) transplanted into the liver of a recipient animal using anti-GFP antibody. Cyclin D1 double staining in blue. Image from verified customer review.

Total cell lysates (20ug) with protein of interest tagged with GFP was loaded. Blotted with GFP antibody [HRP] NB600-308H, without any secondary antibody. Image from verified customer review.

Germaria from wt or chico1/KG mutant ovaries endogenously expressing Myc-GFP stained with anti-GFP, anti-1B1, and DAPI. Scale bars, 10 um. (F) Quantification of relative Myc-GFP intensity in germarium region 2B from wt or chico1/KG mutant ovaries. n = 10 germaria for each genotype. Error bars represent SEM. *p<0.005. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31612862/) licensed under a CC-BY license.

Western Blot of GFP antibody. Lane 1: Wild type GFP (0.1 ug) was used to spike HeLa whole cell lysate. Lane 2: none. Load: 30 ug per lane.Primary antibody: GFP antibody at 1:1000 for overnight at 4C.Secondary antibody: IRDye800(TM) Goat-a-Rabbit IgG [H&L] MX10 at 1:10,000 for 45 min at RT.Block: 5% BLOTTO in PBS overnight at 4C.Predicted/Observed size: 27 kDa for epitope tag GFP. Other band(s): none.

Western Blot of GFP antibody. Lane 1: 293FT cells transfected with CDK4 dominant negative (C-).Lane 2: 293FT cells positive control (C+).Load: 25 ug per lane.Primary antibody: GFP antibody at 1:400 for overnight at 4C.Secondary antibody: IRDye800(TM) rabbit secondary antibody at 1:10,000 for 45 min at RT.Block: 5% BLOTTO overnight at 4C.Predicted/Observed size: 27 kDa for GFP.

Western Blot of GFP antibody. Marker: Opal Pre-stained ladder. Lane 1: HEK293 lysate. Lane 2: HeLa Lysate. Lane 3: CHO/K1 Lysate. Lane 4: MDA-MB-231. Lane 5: A431 Lysate. Lane 6: Jurkat Lysate. Lane 7: NIH/3T3 Lysate. Lane 8: E-coli HCP Control. Lane 9: FLAG Positive Control Lysate. Lane 10: Red Fluorescent Protein. Lane 11: Green Fluorescent Protein. Lane 12: Glutathione-S-Transferase Protein. Lane 13: Maltose Binding Protein. Load: 10 ug of lysate or 50ng of purified protein per lane. Primary antibody: GFP antibody at 1ug/mL overnight at 4C. Secondary antibody: Peroxidase rabbit secondary antibody at 1:30,000 for 60 min at RT. Blocking Buffer: 1% Casein-TTBS for 30 min at RT.Predicted/Observed size: 30 kDa for GFP.

Adult mouse brain tissue. This image was submitted via customer review.

Immuno-microscopy of GFP antibody. Monocyte derived dendritic cells and dermal macrophages were challenged and directly visualized with eGFP labeled Dengue virus to localize sequestration of virus particles in the different cells (upper). The location of the GFP was confirmed by TEM (lower magnified view) using rabbit anti GFP Primary antibody (1:200) and a gold labeled secondary antibody.

Analysis of GFP in mouse retinal cells. Image courtesy of anonymous customer review.

IP analysis of GFP in S2 Cells (Drosophila). Image from verified customer review.

Immunoprecipitation experiments showing that GFP-MISP binds endogenous IQGAP1 (upper panel) and MISP binds to IQGAP1 endogenously using specific antibodies (lower panel) in mitotically arrested (taxol and nocodazole, respectively) HeLa cell lysates. Image collected and cropped by CiteAb from the following publication (nature.com/articles/s41598-018-24682-8), licensed under a CC-BY license.

Immunocytochemistry/ Immunofluorescence: GFP Antibody [NB600-308] - Myc is post-transcriptionally regulated by the IIS-GSK3 cascade.(A) Germaria from ovaries of ctrl, chicoRNAi, & bskRNAi driven by nos-Gal4 expressing LacZ under the control of myc endogenous promoter stained with anti-beta -galactosidase & anti-1B1. Scale bars, 10 μm. (B) A representative live imaging of germaria from flies expressing GFP-tagged Sgg under its endogenous promoter. Scale bar, 10 μm. (C) A germarium from wt flies endogenously expressing Myc-GFP stained with anti-GFP (green) & anti-p-GSK3 (red). Germarium regions are indicated. Myc protein highly corresponds to inhibited form of GSK3. Scale bar, 10 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31612862), licensed under a CC0-1.0 license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: GFP Antibody [NB600-308] - Myc protein pattern in the ovary.(A) A representative low-magnification image of ovarioles from flies endogenously expressing Myc-GFP stained with anti-GFP, anti-1B1, & DAPI. Note that Myc level is low in early germarium stages, becomes high from germarium region 2B, & reduces from the stage-10 egg chamber. Scale bar, 50 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31612862), licensed under a CC0-1.0 license. Not internally tested by Novus Biologicals.

Western Blot: GFP Antibody [NB600-308] - MISP interacts & co-localizes with IQGAP1. (a) HEK293T cells transiently overexpressing GFP-IQGAP1 & FLAG-MISP were used for co-immunoprecipitation experiments from both sides (GFP trap or FLAG M2 beads). (b) Endogenous immunoprecipitation using IQGAP1 antibody or control immunoglobulin (IgG) was carried out in HeLa cell lysates & MISP was detected in the eluate. (c) In vitro interaction between purified MBP-MISP & GST-IQGAP1 was detected by GST & MBP pull-down experiments. (d) Immunoprecipiation experiments showing that GFP-MISP binds endogenous IQGAP1 (upper panel) & MISP binds to IQGAP1 endogenously using specific antibodies (lower panel) in mitotically arrested (taxol & nocodazole, respectively) HeLa cell lysates. (a–d) Images were gained from the same Western blot membrane after cutting/cropping & presented with different exposure times or contrast enhancement for better presentation purposes. The dividing lane marks the grouping of images of the same (or different) membrane. Uncropped blots, where applicable, are included in Supplementary Fig. 4. (e) HeLa cells were immunostained for MISP & IQGAP1 & co-localization was visualized in mitosis in single-plane confocal images, scale bar: 5 μm. Lower pictures: After manual thresholding white spots mark the co-localizing areas in the merge images. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29679050), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: GFP Antibody [NB600-308] - BRC-1 & BRD-1 are inter-dependent for localization.A) Co-localization between BRD-1::GFP (green) & TagRFP-T::BRC-1 (red) at late pachytene in live worms. Scale bar = 10 μm. B) Stills of germline nuclei from live worms expressing GFP::BRC-1 & mCherry::Histone H2B (mCherry::his-58; gfp::brc-1); merge & GFP channel; top two panels, respectively. GFP::BRC-1 expression in brd-1(ok1623) mutant at indicated meiotic stages. Bottom two panels show BRD-1::GFP localization in wild type & the brc-1(xoe4) mutant. Images are projections through half of the gonad. TZ = transition zone; EP = early pachytene; MP = mid pachytene; LP = late pachytene; DP = diplotene; DK = diakinesis. Scale bar = 5 μm. C) Immunoblot of whole worm extracts from indicated worms probed with anti-GFP & alpha -tubulin antibodies. Lane 1 = N2: wild type; Lane 2 = JEL515: gfp::brc-1; Lane 3 = JEL520: gfp::brc-1 brd-1(ok1623); Lane 4 = JEL744: brc-1(xoe4) brd-1::gfp; Lane 5 = JEL657: brd-1::gfp; Lane 6 = JEL678: brc-1(tm1145) brd-1::gfp. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30383767), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GFP Antibody [NB600-308] - BRC-1 & BRD-1 are inter-dependent for localization.A) Co-localization between BRD-1::GFP (green) & TagRFP-T::BRC-1 (red) at late pachytene in live worms. Scale bar = 10 μm. B) Stills of germline nuclei from live worms expressing GFP::BRC-1 & mCherry::Histone H2B (mCherry::his-58; gfp::brc-1); merge & GFP channel; top two panels, respectively. GFP::BRC-1 expression in brd-1(ok1623) mutant at indicated meiotic stages. Bottom two panels show BRD-1::GFP localization in wild type & the brc-1(xoe4) mutant. Images are projections through half of the gonad. TZ = transition zone; EP = early pachytene; MP = mid pachytene; LP = late pachytene; DP = diplotene; DK = diakinesis. Scale bar = 5 μm. C) Immunoblot of whole worm extracts from indicated worms probed with anti-GFP & alpha -tubulin antibodies. Lane 1 = N2: wild type; Lane 2 = JEL515: gfp::brc-1; Lane 3 = JEL520: gfp::brc-1 brd-1(ok1623); Lane 4 = JEL744: brc-1(xoe4) brd-1::gfp; Lane 5 = JEL657: brd-1::gfp; Lane 6 = JEL678: brc-1(tm1145) brd-1::gfp. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30383767), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: GFP Antibody [NB600-308] - Other IIS components are not altered by bsk RNAi.(A) Confocal images for germaria expressing Chico-GFP, myc-Dp110 or PDK1-GFP in the background of ctrl or bskRNAi from ovaries stained with anti-GFP or anti-myc. Scale bars, 10 μm. (B) Ovaries expressing LacZ driven by the puc promoter stained with anti-beta -galactosidase, anti-1B1, & DAPI. Note that JNK activity is moderately induced in late germarium & decreased in the following stages, while JNK activity is much stronger in follicle cells of a maturing egg. Scale bars, 20 μm. (C) Germaria from ovaries of ctrl & bskRNAi driven by nos-Gal4 stained with anti-AKT & anti-1B1. Scale bars, 10 μm. (D) Quantification of AKT intensity from cysts in germarium region 2A & 2B of ovaries with ctrl & bskRNAi driven by nos-Gal4. Intensities are normalized to the value of ctrl at region 2B. n = 7 & 8 germaria for ctrl & bskRNAi, respectively. Error bars represent SEM. *p<0.005. (E) Visualization of the InR mRNA by FISH in germaria from ovaries of ctrl & bskRNAi driven by nos-Gal4. Note that InR mRNA level is decreased by bsk RNAi. Germaria are outlined with dotted lines. Scale bars, 10 μm. (F) Quantification of InR mRNA density in region 2A & 2B germ cells from ovaries with indicated genotypes. n = 9 germaria for each genotype. Error bars represent SEM. *p<0.005. (G) Quantification of relative Myc-GFP intensity in region 2B cysts of ovaries illustrated in Figure 5H. n = 10 germaria for each genotype. Error bars represent SEM. *p<0.005.10.7554/eLife.49309.017Figure 5—figure supplement 1—source data 1.Relative AKT intensity, InR mRNA density, & Myc-GFP intensity in the germarium.Relative AKT intensity, InR mRNA density, & Myc-GFP intensity in the germarium. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31612862), licensed under a CC0-1.0 license. Not internally tested by Novus Biologicals.

$Western Blot: GFP Antibody [NB600-308] - GFP Expression within A. thaliana upon infiltration with SWNT-PM-CytKH9/pDNA complexes.a Reporter construct design of transient GFP expression for the nucleus & mitochondria. b Representative confocal laser scanning microscopy images of A. thaliana root cells from a minimum of five seedlings for each condition (n = 5) 18 h post infiltration with SWNT-PM-CytKH9/pDNA complexes containing pDONR-35S-GFP or pDONR-Cox2-GFP reporter constructs for nuclear or mitochondrial expression, respectively. Mitochondria were stained with MitoTracker Red (CMXRos), & colocalization analysis was performed on GFP expression & MitoTracker signals. Scale bars represent 20 μm. c, d Representative western blots & their quantification from cytosolic (c) & mitochondrial (d) fractions isolated from approximately 60 seedlings 18 h post infiltration with SWNT NCs probed for GFP expression. Actin & cytochrome c were used as loading controls for cytosolic & mitochondrial proteins, respectively. Data points from five biological replicates (n = 5) are represented as the mean ± standard deviation. Statistical significance was determined by Brown-Forsythe & Welch one-way ANOVA test. For the cytosolic samples, P-values are 0.0027, & 0.0020 between DNA only & SWNT-PM-CytKH9, SWNT-PM-Cytcox, respectively, & 0.0253 between SWNT-PM-CytKH9 & SWNT-PM-Cytcox. For the mitochondrial samples, P-values are 0.0019, & 0.0333 between DNA only & SWNT-PM-CytKH9, & SWNT-PM-Cytcox, respectively, & 0.0359 between SWNT-PM-CytKH9 & SWNT-PM-Cytcox. ns – not statistically significant, *P < 0.05, **P < 0.01. Source data are provided as a Source Data file. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35577779), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: GFP Antibody [NB600-308] - VGLL1 regulates MMP9 transcription in gastric cancer cells. (a,b) Viability (a) & invasion (b) assay of NUGC3 cells. Cells were treated with 20 nM siScramble (SC) or siMMP9 for 48 h, & then stained with sulforhodamine B. Data are presented as mean ±SD. n = 3; * p < 0.05 (Student’s t-test). (c) MMP9 mRNA expression regulation by VGLL1 in gastric cancer cells treated with siVGLL1 or siSC (control) was measured by qPCR. (d) Changes in MMP9 expression upon knockdown or overexpression of VGLL1 assayed by western blotting in NUGC3 cells. (e) IHC of VGLL1 & MMP9 expression in the liver & lungs of an in vivo metastasis mouse model using surgical resection of tumors. Scale bar, 200 µm. (f) Construction of various luciferase reporter systems under control of the MMP9 promoter. (g) MMP9 promoter activities of the reporter systems containing modified TEA-binding sites were measured in NUGC3 cells. n = 3; * p < 0.05 (Student’s t-test). (h) Effect of TEAD4 on VGLL1-regulated MMP9 transcriptional activity. NUGC3 cells treated with siSC or siTEAD4 for 24 h were transfected with MMP9-luc, Renilla-luc, pCDNA3.1, & pcDNA3.1-myc-VGLL1 vectors for 48 h. n = 3; * p < 0.05 (Student’s t-test). (i) Interaction between VGLL1 & TEAD4. Lysates of NUGC3 cells that were transfected with pcDNA3.1-myc-VGLL1 & pEGFP-N1-TEAD4 were immunoprecipitated using anti-IgG, anti-GFP, & anti-Myc antibodies. Protein expression was analyzed by immunoblotting. (j) ChIP assays while using nuclear extracts of NUGC3 cells treated with siTEAD4. The ChIP-enriched DNA was subjected to PCR. (k) Target genes of VGLL1 & YAP. NUGC3 cells were transfected with pcDNA3, pcDNA3-myc-VGLL1, or pcDNA3-myc-YAP, & the mRNA expression levels were analyzed by RT-PCR. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31816819), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/Immunofluorescence: Rabbit GFP pAb [NB600-308] - Analysis of GFP in human Hs746T gastric cancer cell line. Image from a verified customer review.

Reactivity:

All-NA

Species Glossary

Reactivity: All-NA

Applications:

, , , , , , , , ,

Applications: , , , , , , , , ,

Host:

Rabbit Polyclonal

Host: Rabbit Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Conjugate	Catalog #	Availability	Size	Price
Alexa Fluor 350	NB600-308AF350
Alexa Fluor 405	NB600-308AF405
Alexa Fluor 488	NB600-308AF488
Alexa Fluor 532	NB600-308AF532
Alexa Fluor 594	NB600-308AF594
Alexa Fluor 647	NB600-308AF647
Alexa Fluor 700	NB600-308AF700
Alexa Fluor 750	NB600-308AF750
Allophycocyanin	NB600-308APC
Allophycocyanin/Cy7	NB600-308APCCY7
Biotin	NB600-308B
CoraFluor 1	NB600-308CL1
DyLight 350	NB600-308UV
DyLight 405	NB600-308V
DyLight 488	NB600-308G
DyLight 550	NB600-308R
DyLight 594	NB600-308DL594
DyLight 650	NB600-308C
DyLight 680	NB600-308FR
DyLight 755	NB600-308IR
FITC	NB600-308F
HRP	NB600-308H
Janelia Fluor 525	NB600-308JF525
Janelia Fluor 549	NB600-308JF549
Janelia Fluor 646	NB600-308JF646
Janelia Fluor 669	NB600-308JF669
PE	NB600-308PE
PE/Atto594	NB600-308PEATT594
PE/Cy5.5	NB600-308PECY55
PE/Cy7	NB600-308PECY7
PerCP	NB600-308PCP
mFluor Violet 450 SE	NB600-308MFV450
mFluor Violet 500 SE	NB600-308MFV500
mFluor Violet 610 SE	NB600-308MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB600-308-1mg
BSA Free	NB600-308SS

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AF1924

SOX17 Antibody [Unconjugated]

(3 Reviews)

(506 Publications)

100 μg

AF1924

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.

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 (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.

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; NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

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; NL001) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

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 # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

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 # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

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 # 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.

Generation and characterization of systemic lupus erythematosus (SLE)‐specific human‐induced pluripotent stem cells (hiPSCs). Immunostaining of Otx2 (ectoderm marker), Sox17 (endoderm marker), Brachyury (mesoderm marker) and co‐staining with DAPI (blue) in embryoid bodies (EBs) derived from hiPSCs‐SLE. Scale bar, 50 μm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31536674), licensed under a CC-BY licence.

Concordant expression of developmental programs across organoids from human iPSC lines. 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 licence.

Activin-A levels during Day 0–1 modulate cardiomyocyte (CM) vs. definitive endoderm (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. 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. f-i shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. Scale bar: 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 licence.

In vitro and in-TiPSCs. Immunofluorescence staining for Sox17 (endodermal marker), alpha SMA (mesodermal marker), and Nestin (ectodermal marker) in each human TiPSCs1-derived differentiated cell in vitro. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0097397), licensed under a CC-BY licence.

Differentiation of hESCs into definitive endoderm cells by Activin A with CHIR99021. Immunofluorescence analysis of the expression of SOX17 and FoxA2 for Activin A with 2 µM CHIR99021-induced differentiated cells on day 3. 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 licence.

HIF-1 alpha activates transcription of Sox17. Western blot analysis in control human lung microvascular endothelial cells (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. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY licence.

Features of ESC-INTPSCs intermediate between ESCs and EpiSCs. Expression of OCT3/4 and SOX17. Scale bar, 100 µm. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0095329), licensed under a CC-BY licence.

p38-Mapk14/11 inhibition during murine blastocyst maturation blocks primitive endoderm (PrE) differentiation/maturation. 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. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27605380), licensed under a CC-BY licence.

Endothelial cell (EC) specific deletion of Sox17 (Sox17EC−/−) in mice prevents endothelial regeneration. Western blot analysis of Sox17 protein expression in isolated ECs obtained from flushed lungs of Sox17EC−/− and control mice. n = 4. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY licence.

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‐SLE clones by Student's t test showing P‐values ≤ .05 in each comparison. (I) Immunostaining of Otx2 (ectoderm marker), Sox17 (endoderm marker), Brachyury (mesoderm marker) and co‐staining with DAPI (blue) in EBs derived from hiPSCs‐SLE. Scale bar, 50 μm Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31536674), licensed under a CC-BY license. Not internally tested by R&D Systems.

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.

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.

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 DAPI. At day 3 post-LPS, the control group showed a significantly higher number of BrdU+ECs compared to baseline. However, Sox17EC−/− mice showed markedly reduced level of BrdU+ECs, indicating reduced EC proliferation. i To assess whether expression of Sox17 in ECs can restore lung endothelial integrity, studies were performed in Sox17EC−/− mice to overexpress Sox17 protein. We used a mixture of 50 μg plasmid (mouse Cdh5 promoter—Flag —Sox17) encapsulated in liposomes, which were injected i.v. 3 h after LPS challenge. j At day 3 post-LPS, liposome vector-treated Sox17EC−/− mice showed marked EC barrier leakiness as assessed by lung transvascular permeability of albumin whereas the Sox17-rescued mice showed markedly reduced endothelial permeability. n = 4. OE, overexpression. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-tailed Student’s t-test for (c, j), two-way ANOVA with Bonferroni post-tests for (d, e, g, h) and Log-rank (Mantel-Cox) test for (f) 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.

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 cells positively immunofluorescently staining for both EPI and PrE ICM markers using anti-Nanog and anti-Gata6 (an early PrE marker) antibodies. All individual embryo data used in the preparation of this figure are contained within the electronic supplementary material, table S3. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27605380), licensed under a CC-BY license. Not internally tested by R&D Systems.

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.

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.

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Reactivity: Hu

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Allophycocyanin	IC1924A
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Carrier Free	IC1924A
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NB600-1384

LC3B Antibody - BSA Free

(19 Reviews)

(448 Publications)

0.1 ml

NB600-1384

FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA and then pre-treated for 1 h with pepstatin A (PA, 10 uM) and E64D (10 uM) as indicated. Cells were subsequently treated for 3 h with HBSS in continuous presence or absence PA/E64D inhibitors. LC3-I/II levels were assayed by Western blot. Image collected and cropped by CiteAb from the following publication (//jeccr.biomedcentral.com/articles/10.1186/s13046-019-1328-3) licensed under a CC-BY license.

LC3B immunohistochemical staining in CC tissue(A) CC with low LC3B dot like staining. (B) CC with high LC3B dot like staining. (magnification 20X). Image collected and cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.17554) licensed under a CC-BY license.

Analysis of LC3B in treated U87-MG (human glioblastoma astrocytoma) lysates using anti- [Catalog # NB600-1384].

Depletion of RARalpha prevents ATRA-induced autophagic flux in SKBR3 cells. LC3B western blotting and quantification of the autophagic activity in control and RARalpha-knockdown SKBR3 cells upon treatment with 1 uM ATRA for 2 days in the presence or absence of BafA for 2 h at 200 nM. LC3B-II expression was normalized to GAPDH and to vehicle-treated control cells. Standard deviations from at least five independent experiments are shown. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2015236), licensed under a CC-BY license.

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>Fibroblasts were exposed to 1 mM LLOMe and when indicated pretreated with the autophagy inhibitors 3-methyladenine (5 mM, 1 h) or bafilomycin A1 (25 nM, 30 min). Immunostaining with corresponding colocalization analysis of a) LC3 (green) and galectin-3 (red) and b) LC3 (green) and LAMP2 (red). Merged images show colocalization in yellow, nuclei are stained with DAPI (blue). LLOME = l-leucyl-l-leucine methyl ester, a lysosomal membrane damage compound. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32409651/) licensed under a CC-BY license.

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).

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

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.

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.

LC3A and LC3B siRNAs specifically block the expression of the LC3A and LC3B proteins, respectively, in A549 cell line (E1,2,3). In E4 the reactivity of and of the LC3B (5F10 antibody) is shown, following silencing of the LC3A or of the LC3B genes, in the A549 cell line. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0137675), licensed under a CC-BY license.

NOD1-dependent induction of autophagy contributes to IRGM and LC3 recruitment to Mtb-containing vesicles. The subcellular localization of autophagy proteins was observed in untreated (A, C) and Tri-DAP treated cells (B, D) using anti-IRGM and anti-LC3 antibodies, which were detected using a secondary antibody coupled to 5 nm gold particles (indicated with arrowheads; TEM X 62,000). Gold particles colocalizing with bacteria were manually counted in 10 macrophages of each condition (E, F) and the differences between the treatments are indicated: ***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 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 0.01); (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); (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 0.01, **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); (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) 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); (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); (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] - 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 > 0.01); (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); (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) 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] - 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 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] - 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] - 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
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)

(448 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.

Application of the approach to other cell lines. The remainder of the protocol from Figure 2 was then applied using 100 ug/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 and probing with antibodies to GAPDH, BiP and Histone H3 (Used as an alternative nuclear marker due to weakness of the Lamin A signal in HeLa cells). Image collected and cropped by CiteAb from the following publication (//bmcresnotes.biomedcentral.com/articles/10.1186/1756-0500-2-243) licensed under a CC-BY license.

Endothelial responses provoked by Hb-lipid interactions are attenuated by sulfide donors. Confluence HAoECs were exposed to the obtained Hb samples for 8 hours and VCAM-1 expression was determined by Western blotting. GAPDH were used as a loading control. Representative experiment, n = 3. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/omcl/2018/3812568/), licensed under a CC-BY license.

Regulation of cell migration-associated proteins by NCKU-21 in A549 and CL1-5 cells. Protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 were analyzed in A549 cell treated with NCKU-21 for 24 hr. * P 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] - 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] - 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] - 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.

$Western Blot: GAPDH Antibody (1D4) [NB300-221] - Role of the TRIM11-USP14 axis in cell growth & apoptosis. a Levels of endogenous TRIM11 in HCT116 cells grown at 43 °C for 0 or 90 min, or at 43 °C for 90 min, & then at 37 °C for 60 or 120 min. b, c Levels of endogenous TRIM11 (b), or Flag-TRIM11, the indicated proteasome subunits, & USP14 (c) in the SN & P fractions of HCT116 cells with & without heat shock at 43 °C for 90 min. d, g TRIM11 overexpression (d) or knockdown (g) HCT116 cells & the corresponding control cells grown under unstressed, heat shock, & heat shock plus recovery conditions were analyzed for K48 polyUb conjugates & caspase-3 activation. e, f, h Apoptosis in TRIM11 overexpression (e, f) or knockdown (h) HCT116 cells, & the corresponding control HCT116 cells, treated with & without heat shock. i, j Caspase-3 activation (i) & apoptosis (j) in HCT116 cells expressing the indicated proteins and grown under normal or heat stress conditions. k–m K48 polyUb conjugates, caspase-3 activation (k), amyloid-like fibrils (l), & apoptosis (m) in HCT116 cells expressing the indicated proteins and grown under normal & heat stress conditions. In e, f, h, j, l, & m, data represent mean ± SEM (n = 3 unless otherwise indicated). *P < 0.05; **P < 0.01; ***P < 0.001. Uncropped blots are presented in Supplementary Fig. 14 Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-018-03499-z), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Reactivity:

Hu, Mu, Rt, Po, Av, Bv, Ca, Ch, ChHa, Dr, Eq, Fe, Ha, Ma-Op, Pm, Rb, Sh, Ze

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Av, Bv, Ca, Ch, ChHa, Dr, Eq, Fe, Ha, Ma-Op, Pm, Rb, Sh, Ze

Applications:

, , , , , , ,

Applications: , , , , , , ,

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

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Unconjugated

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BSA Free	NB300-221SS

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H00008878-M01

p62/SQSTM1 Antibody (2C11) - Azide and BSA Free

(9 Reviews)

(433 Publications)

0.1 mg

H00008878-M01

Immunocytochemistry/Immunofluorescence: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - ICC-IF analysis of SQSTM1 protein in HeLa cell using SQSTM1/p62 antibody (clone 2C11) at 10 ug/ml concentration.

Immunocytochemistry/Immunofluorescence: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Analysis of p62 in SH-SY5Y cells using anti-p62/SQSTM1 antibody. Red - p62 puncta; Blue - nuclear DAPI; Green - Cytoskeleton. Image from verified customer review.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Disruption of the VANGL2-p62/SQSTM1 interaction in breast cancer cells. Soft agar colony formation of T47D cells overexpressing GFP, GFP-VANGL2 and GFP-p62/SQSTM1 (right). Protein expression was revealed with anti-p62/SQSTM1, anti-VANGL2 and anti-GFP antibodies by western blot analysis (right). In anti-GFP blot, the arrowhead indicates position of co-migrating GFP-VANGL2 and GFP-p62/SQSTM1 and the asterisk pinpoints GFP alone. Error bars represent mean+/-s.d. (n=3). Image collected and cropped by CiteAb from the following publication (//www.nature.com/doifinder/10.1038/ncomms10318), licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Analysis of p62/SQSTM1 antibody on HeLa cells. Image from verified customer review.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Confirmation of loss of hepatic ATG7 protein and a phenotype of hepatic autophagy inactivation. Fourth ug of liver protein were loaded on 4-20% gradient Tris-Glycine gels and subsequently immunoblotted with antibodies against ATG7, p62 and LC3. Blots were visualized with G:BOX Chemi XRQ (SynGene). Representative immunoblots are shown for WT and KO mice. Full-length immunoblots are presented in Supplementary Figure S1. ATG7: autophagy-related protein 7; p62: sequestosome 1; LC3: microtubule-associated protein 1 light chain 3; WT: Mx1-Cre-Atg7F/F mouse; KO: Mx1-Cre+Atg7F/F mouse. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-017-14405-w), licensed under a CC-BY license.

ELISA: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Detection limit for recombinant GST tagged SQSTM1 is approximately 0.03ng/ml as a capture antibody.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Activation of multiple signaling pathways by dandelion root extractA. Western blots of proteins involved in programmed cell death & cell survival & inflammation B. Densitometry quantification of western blot analysis from three independent experiments. *P<0.05, **P< 0.001. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.11485), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Elevated levels of additional proteins & GM3 ganglioside in the MEC of MPS IIIB brain.Staining performed with antibodies to the indicated substances was observed in the MEC region of 3 month-old MPS IIIB mice (for total ubiquitin & polyubiquitin) & 6 months for all others. Staining was not seen in the MEC region of age-matched control mice (Naglu +/−) nor in the LEC region of MPS IIIB mice (the latter not shown). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0027461), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Confirmation of loss of hepatic ATG7 protein & a phenotype of hepatic autophagy inactivation. Fourty µg of liver protein were loaded on 4–20% gradient Tris-Glycine gels & subsequently immunoblotted with antibodies against ATG7, p62 & LC3. Blots were visualised with G:BOX Chemi XRQ (SynGene). Representative immunoblots are shown for WT & KO mice. Full-length immunoblots are presented in Supplementary Figure S1. ATG7: autophagy-related protein 7; p62: sequestosome 1; LC3: microtubule-associated protein 1 light chain 3; WT: Mx1-Cre−Atg7F/F mouse; KO: Mx1-Cre+Atg7F/F mouse. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29074879), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

$Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - The content of mitochondrial p62, OPTN, & VCP in spinal cord of SOD1‐G93A mice is decreased by Parkin knockoutImmunostaining of anterior horn of lumbar spinal cord for ChAT (in green) & p62 (in red) at 130 days. Scale bar, 10 μm.Quantification of the percentage of ChAT‐positive motor neurons (MN) containing p62‐positive inclusions at 130 days. PKO/G93A MN have less SOD1 aggregates than G93A, while Non Tg & PKO MN have none. Data were collected from n = 4 (two males & two females) mice for Non Tg & PKO & n = 6 (three males & three females) for G93A & PKO/G93A. Number of images recorded for each genotype: n = 9 for Non Tg & PKO, n = 16 for G93A, & n = 12 for PKO/G93A. Results are expressed as mean ± SEM; *P = 0.032, by unpaired one‐tailed Student's t‐test.Representative Western blots of OPTN, p62, & VCP in spinal cord mitochondrial fractions at 130 days. Protein levels are normalized by Complex V. The quantifications in panels (D–F) show that mitochondria of PKO/G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V.Quantification of OPTN relative to Complex V at 130 days. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females); *P = 0.039 by paired Student's t‐test.Quantification of p62, relative to Complex V at 130 days. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females); **P = 0.0029 by paired Student's t‐test.VCP was quantified using Complex V as loading reference at 130 days. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females); *P = 0.044 by paired Student's t‐test.Source data are available online for this figure. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30126943), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - miR-29a downregulates TFEB & ATG9A to inhibit autophagyA. Schematic representation of the miR-29 family members & 3′-UTR binding sites of miR-29 targets as well as mutated binding sites used in Luciferase Assays: Transcription Factor EB (TFEB) & Autophagy-related protein 9A (ATG9A). All three miR-29 family members (miR-29a, miR-29b, & miR-29c) have identical seed sequences. Conserved miR-29 binding sites in the 3′-UTR of mRNA transcripts encoding ATG9A & TFEB are depicted in bold. B. 10ug of total protein cell lysates from Panc-1 transfected with CTRL or miR-29a mimics were subjected to western blot analysis for TFEB, ATG9A, & GAPDH. Relative quantification of band intensities normalized to GAPDH are shown below respective blots. C. Relative firefly luciferase activity from TFEB & ATG9A 3′ UTR wild type (WT) & mutant (mut) reporter constructs following co-transfection into Panc-1 cells with control or miR-29a mimics. All readouts were normalized to renilla luciferase activity for each well. Average relative luminesce normalized to respective controls is presented (n=6) ± S.E.M. D. 5ug of total protein cell lysates from Panc-1 cells were transfected with CTRL, miR-29a mimics, siCTRL, siTFEB, or siATG9A. 24 hours post-transfection, total protein was harvested & subjected to western blot analysis for p62 & LC3B, & GAPDH was used as loading control. Quantification of band intensities normalized to GAPDH & relative to control are shown below respective blots. All experiments were repeated 3 times & representative data is presented. Image collected & cropped by CiteAb from the following publication (//www.oncotarget.com/lookup/doi/10.18632/oncotarget.11928), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Effects of autophagy inhibitors in combination with IFN-gamma on expression of PD-L1 in gastric cancer cell lines. a The effect of chloroquine (CQ) or 3-MA on expression of PD-L1 with or without INF-gamma for 24 h was determined by flow cytometry assays. In AGS & NCI-n87 cells, MFI as the indication of PD-L1 expression level can be further increased by the treatment of INF-gamma . b Levels of LC3B-I/II, p62/SQSTM1 & PD-L1 were determined by Western blots in AGS & NCI-n87 cells treated by CQ, 3-MA, bafilomycin A1 (Baf) or rapamycin (Rap) for 24 h. c Positive staining of PD-L1 (red) & LC3 positive puncta (green) was determined by immunofluorescence in AGS & NCI-n87 cells treated by autophagy inhibitors & activator as in (b). d Rapamycin decreased the levels of PD-L1 protein in AGS & NCI-n87 cells as shown by flow cytometry. Results were averaged & blots were representative of 4 independent experiments, *p < 0.05, **p < 0.01 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.

$Immunocytochemistry/ Immunofluorescence: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - The content of mitochondrial p62, OPTN, & VCP in spinal cord of SOD1‐G93A mice is decreased by Parkin knockoutImmunostaining of anterior horn of lumbar spinal cord for ChAT (in green) & p62 (in red) at 130 days. Scale bar, 10 μm.Quantification of the percentage of ChAT‐positive motor neurons (MN) containing p62‐positive inclusions at 130 days. PKO/G93A MN have less SOD1 aggregates than G93A, while Non Tg & PKO MN have none. Data were collected from n = 4 (two males & two females) mice for Non Tg & PKO & n = 6 (three males & three females) for G93A & PKO/G93A. Number of images recorded for each genotype: n = 9 for Non Tg & PKO, n = 16 for G93A, & n = 12 for PKO/G93A. Results are expressed as mean ± SEM; *P = 0.032, by unpaired one‐tailed Student's t‐test.Representative Western blots of OPTN, p62, & VCP in spinal cord mitochondrial fractions at 130 days. Protein levels are normalized by Complex V. The quantifications in panels (D–F) show that mitochondria of PKO/G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V.Quantification of OPTN relative to Complex V at 130 days. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females); *P = 0.039 by paired Student's t‐test.Quantification of p62, relative to Complex V at 130 days. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females); **P = 0.0029 by paired Student's t‐test.VCP was quantified using Complex V as loading reference at 130 days. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females); *P = 0.044 by paired Student's t‐test.Source data are available online for this figure. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30126943), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

$Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - G2019S LRRK2 hippocampal neurons do not have elevated alpha -synuclein pathology 14 days post-transduction. a Primary hippocampal neurons from NTG or G2019S pups were transduced with 2.5 μg/mL alpha -synuclein PFFs & allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble alpha -synuclein & p62 are similar in both neuron types. b Quantification of soluble proteins shows ~ 25-fold elevation in the expression of LRRK2 & a commensurate ~ 50-fold elevation in pS395 LRRK2, indicative of the elevated LRRK2 kinase activity associated with the G2019S mutation. Soluble alpha -synuclein levels were equivalent between the cultures. c No significant differences were found between the genotypes in insoluble proteins by an unpaired t-test with Welch’s correction. (N = 3 biological replicates for each protein). Means + s.e.m.; **P < 0.01; *P < 0.05 by an unpaired t-test with Welch’s correction for unequal variances. All values are normalized to NTG neurons treated with alpha -synuclein PFFs & DMSO Image collected & cropped by CiteAb from the following publication (//actaneurocomms.biomedcentral.com/articles/10.1186/s40478-018-0550-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

$Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - LRRK2 inhibition does not reduce insoluble alpha -synuclein in wildtype hippocampal neurons. a Primary hippocampal neurons from CD1 pups were treated with 30 nM LRRK2 inhibitors PF-475, PF-360 or DMSO as a vehicle control, then transduced with 2.5 μg/mL alpha -synuclein PFFs & allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble alpha -synuclein & p62 are similar in both LRRK2 inhibitor treated & untreated neurons. b Quantification of soluble proteins show some reduction of LRRK2 protein levels PF-475 treatment & ~ 75% inhibition of LRRK2 activity (as assayed by pS935 LRRK2) by both PF-475 & PF-360. c Insoluble pS129 alpha -synuclein was slightly, but significantly elevated by PF-360 treatment, while alpha -synuclein & p62 were unchanged by one-way ANOVA. (N = 5 biological replicates for each protein). Means + s.e.m.; *P < 0.05, ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. All values are normalized first to GAPDH, as a loading control, then to DMSO-treated neurons Image collected & cropped by CiteAb from the following publication (//actaneurocomms.biomedcentral.com/articles/10.1186/s40478-018-0550-0), licensed under a CC-BY license. Not internally tested by Novus Biologicals.$

Immunocytochemistry/ Immunofluorescence: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Colocalization of VANGL2 & p62/SQSTM1 in late endosomes.(a) Immunofluorescence staining of SKBR7 cells showed colocalization of endogenous VANGL2 (green) & p62/SQSTM1 (red) in discrete cytoplasmic puncta. Scale bar, 10 μm. The mean Pearson correlation for VANGL2 & p62/SQSTM1 is 0.62, calculated using the Image J software for ∼15 cells per field of view & from 10 images. (b) Partial colocalization of VANGL2 & p62/SQSTM1 in late endosomes of SKBR7 cells stained w/ the LAMP1 marker. Scale bar, 20 μm. (c) SKBR7 ells cultured in a nutrient-deprived medium & treated w/ 100 nM bafilomycin A1 (6 h) before fixation. Double labelling against VANGL2 (arrowheads) & p62/SQSTM1 (arrows), as described in Methods, showed accumulations of both markers in vesicular structures probably resembling endosomes/amphisomes. Scale bar, 200 nm. (d) IMCD3 cells treated w/ PBS or EGTA (5 mM) for 30 min. Immunofluorescence & confocal analysis performed using the indicated antibodies. Scale bar, 10 μm. Inserts show colocalized VANGL2 & p62/SQSTM1. (e) The VANGL2–p62/SQSTM1 complex recovered in confluent SKBR7 or IMCD3 cells w/ 2G4 mAb (IP VANGL2) but not a control antibody (IP IgG2a ctrl) as seen using western blot analysis w/ the indicated antibodies. The complex more abundant in cancer cells (SKBR7) than in polarized cells (IMCD3). Note that human (SKBR7 cells) & murine (IMCD3 cells) p62/SQSTM1 run at different molecular weights. (f) Lysates of confluent IMCD3 cells treated for 30 min w/ PBS or EGTA subjected to 2G4 mAb immunoprecipitation (IP VANGL2). Immunoprecipitated proteins probed by western blot analysis w/ the indicated antibodies. Increased amounts of VANGL2–p62/SQSTM1 complexes recovered after EGTA treatment. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms10318), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Colocalization of VANGL2 & p62/SQSTM1 in late endosomes.(a) Immunofluorescence staining of SKBR7 cells showed colocalization of endogenous VANGL2 (green) & p62/SQSTM1 (red) in discrete cytoplasmic puncta. Scale bar, 10 μm. The mean Pearson correlation for VANGL2 & p62/SQSTM1 is 0.62, calculated using the Image J software for ∼15 cells per field of view & from 10 images. (b) Partial colocalization of VANGL2 & p62/SQSTM1 in late endosomes of SKBR7 cells stained w/ the LAMP1 marker. Scale bar, 20 μm. (c) SKBR7 ells cultured in a nutrient-deprived medium & treated w/ 100 nM bafilomycin A1 (6 h) before fixation. Double labelling against VANGL2 (arrowheads) & p62/SQSTM1 (arrows), as described in Methods, showed accumulations of both markers in vesicular structures probably resembling endosomes/amphisomes. Scale bar, 200 nm. (d) IMCD3 cells treated w/ PBS or EGTA (5 mM) for 30 min. Immunofluorescence & confocal analysis performed using the indicated antibodies. Scale bar, 10 μm. Inserts show colocalized VANGL2 & p62/SQSTM1. (e) The VANGL2–p62/SQSTM1 complex recovered in confluent SKBR7 or IMCD3 cells w/ 2G4 mAb (IP VANGL2) but not a control antibody (IP IgG2a ctrl) as seen using western blot analysis w/ the indicated antibodies. The complex more abundant in cancer cells (SKBR7) than in polarized cells (IMCD3). Note that human (SKBR7 cells) & murine (IMCD3 cells) p62/SQSTM1 run at different molecular weights. (f) Lysates of confluent IMCD3 cells treated for 30 min w/ PBS or EGTA subjected to 2G4 mAb immunoprecipitation (IP VANGL2). Immunoprecipitated proteins probed by western blot analysis w/ the indicated antibodies. Increased amounts of VANGL2–p62/SQSTM1 complexes recovered after EGTA treatment. Image collected & cropped by CiteAb from the following publication (//www.nature.com/articles/ncomms10318), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Parkin protein levels are unaffected in liver while a small decrease is measured in G93A cerebellumRepresentative Western blot for Parkin & p62 in cerebellum (Cb) homogenates at 130 days.Quantification indicates that Parkin is decreased in G93A mice relative to Non Tg at 130 days. Protein levels were normalized by beta ‐actin. Results are expressed as mean ± SEM & percent of Non Tg; n = 8 (four males & four females) mice per group. *P = 0.039 by paired Wilcoxon's test.Quantification of p62 in the homogenates indicates that p62 is increased in PKO & PKO/G93A mice, independent of SOD1‐G93A expression, at 130 days of age. beta ‐actin was used for normalization. Results are expressed as mean ± SEM & percent of Non Tg; n = 8 (four males & four females) mice per group. No statistically significant differences were found between Non Tg & G93A by paired Friedman's test with Dunn's correction (P = 0.99). ***P = 0.0001 by paired Student's t‐test (G93A vs. PKO/G93A).Representative Western blot of Parkin & p62 in liver homogenates at 130 days.Parkin protein levels were quantified at 130 days. beta ‐actin was used for normalization. Results are expressed as mean ± SEM & percent of Non Tg; n = 8 (four males & four females) mice per group. No statistically significant differences were found between Non Tg & G93A (P = 0.546 by paired Wilcoxon's test).Quantification of p62 protein levels in liver at 130 days. Results are expressed as mean ± SEM & percent of Non Tg; n = 8 (four males & four females) mice per group. No statistically significant differences were found between Non Tg & G93A (P = 0.546 by paired Wilcoxon's test). No statistically significant differences were found among the other groups.Source data are available online for this figure. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30126943), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Parkin knockout mitigates the accumulation of mitophagy adaptors in G93A mitochondria at disease end stageA, BRepresentative Western blots of p62 (A), OPTN, & VCP (B) in spinal cord mitochondria from end‐stage mice.Cp62 quantification, using Complex V as normalizer, shows a strong accumulation of p62 in SOD1‐G93A mitochondria at end stage. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females) mice per group. No statistically significant differences were found between G93A & PKO/G93A (P = 0.078 by paired Wilcoxon's test); ***P = 0.0007 (Non Tg vs. G93A) & *P = 0.037 (PKO & PKO/G93A) both by paired Friedman's test with Dunn's correction.DQuantification of OPTN accumulation in end‐stage mitochondria, with Complex V as protein loading control. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females) mice per group. **P = 0.0078 (for G93A & PKO/G93A) by paired Wilcoxon's test; ***P = 0.0003 (Non Tg vs. G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found.EQuantification of VCP in mitochondria at disease end stage. Complex V was used as normalizer. Results are expressed as mean ± SEM & as percent of Non Tg; n = 8 (four males & four females) mice per group. *P = 0.039 (for G93A & PKO/G93A) by paired Wilcoxon's test; ***P = 0.0007 (Non Tg vs. G93A) & *P = 0.037 (PKO & PKO/G93A) both by paired Friedman's test with Dunn's correction.Source data are available online for this figure. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30126943), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Bv, Ha, Rb

Species Glossary

Reactivity: Hu, Mu, Rt, Bv, Ha, Rb

Applications:

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NB400-105

ABCA1 Antibody - BSA Free

(7 Reviews)

(414 Publications)

0.1 ml

NB400-105

Formalin-fixed paraffin-embedded tissue sections of human prostate cancer were probed for ABCA1 mRNA (ACD RNAScope Probe, catalog # 432291; Fast Red chromogen, ACD catalog # 322360). Adjacent tissue section was processed for immunohistochemistry using rabbit polyclonal (Novus Biologicals catalog # NB400-105) at 1.5ug/mL with overnight incubation at 4 degrees Celsius 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 glandular cells.

<b>Genetic Strategies Validation and Orthogonal Strategies Validation.</b>HSKMCs were transfected with scrambled siRNA (scr siRNA) or ABCA1 siRNA. The cells were lysed and ABCA1 (A) protein levels were quantified by immunoblotting. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41598-018-38014-3) licensed under under a CC-BY license.

<b>Biological Strategies Validation. </b>Analysis of ABCA1 in total cell lysates of RAW264.9 cells treated with vehicle (-) or 9-cisretinoic acid and 22Rhydroxycholesterol (+). Samples used for this testing were 40 ug of total cell post-nuclear lysate from each group.

<b>Biological Strategies Validation. </b>HepG2 cells were grown to 60% confluency, serum starved for 24 hours, and then treated with 1uM TO9 for 24 hours prior to being fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-ABCA1 at 5.0ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. 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 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Untreated HepG2 cells were grown to 60% confluency, and serum starved for 24 hours prior to being fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-ABCA1 at 5.0ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. 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 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

ABCA-1 FITC conjugated antibody of human adipose tissue macrophage subsets by flow cytometry. Image from verified customer review.

Association between expression of ABCA1 and survival in ovarian cancer patients. Expression of ABCA1 in 55 ovarian cancer patient samples was determined by IHC in tissue microarray. Representative image of ovarian cancer showing high (left panel) and low (right panel) ABCA1 expression on the cell membrane or cytoplasm (x400). Image collected and cropped by CiteAb from the following publication (//www.clinicalepigeneticsjournal.com/content/7/1/1), licensed under a CC-BY license.

ABCA1 in human primary fibroblasts in culture. 10 ug of total protein. 7.5% TGX gel. Blocking 5% milk in PBST 1h RT. 1:1000 primary ab in BSA 3% PBST O/N at 4C. Secondary 1:5000 HRP 1h RT. Arrow shows around 250 kDa. WB image submitted by a verified customer review.

ABCA1 antibody was tested at 1: 400 in HeLa cells using an Alexa Fluor 488 secondary (shown in purple). M1 is defined by unstained cells.

<b>Biological Strategies Validation. </b>Detection of ABCA1 in mouse peritoneal macrophages using NB 400-105 (Lot L). ECL exposure, 1 min. Lane 4: T09 uninduced lysate Lane 5: T09 induced lysate.

Prostates of LXR mutant mice accumulate cholesterol esters through inappropriate LXR target genes regulation. Total protein lysates of WT and LXR null mice under normal or high cholesterol diet were analyzed by western blotting with antibodies against ABCA1, LDLR and ACTIN as a loading control (left panel), quantification of ABCA1 and LDLR protein accumulation levels (right panel). * p<0.05, *** p<0.001 in Student's t test. Error bars represent the +/- mean SEM. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pgen.1003483), licensed under a CC-BY license.

miR-33b reduces cellular cholesterol efflux and serum HDL-C levels. Western blotting for ABCA1 (using NB400-105) and ABCG1 proteins in peritoneal macrophages from WT and KI+/+ mice. Representative images are shown. beta-actin was used as the loading control. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep05312), licensed under a CC-BY license.

miR-33b regulates ABCA1 and SREBP-1. Analysis for ABCA1, SREBP-1, CPT1a, and AMPK alpha protein levels in the livers of WT, KI+/-, and KI+/+ mice. Representative images are shown. TF2B and beta-actin were used as loading controls. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep05312), licensed under a CC-BY license.

Detection of ABCA1 in human prostate epithelium showing luminal and membrane staining.

Liver mRNA & protein expression analysis of chow-fed Osbpl8KO mice.A: qPCR analysis of the quantity of the mRNAs identified at the bottom in chow-fed KO females (open bars) & males (closed bars). The mRNAs were quantified using ribosomal protein 36B4 message as a housekeeping reference. The data are expressed relative to quantity in littermate WT animals of the same gender, & represent mean ± s.e.m. (n = 6; *p<0.05, **p<0.01, T-test). B: Western blot analysis of ABCA1 & SR-B1 proteins in WT & KO mouse liver. The blots were probed with anti-beta -actin as a loading control. Densitometric quantification of the Western blot data is shown on the right. The results were normalized against beta -actin. The data represents mean ± s.e.m. (n = 4). Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0058856), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Protein expressions of ABCA1, SR-BI & ABCG1 in liver & macrophage by Western blot. Simvastatin increased the expressions of ABCA1 & ABCG1 in liver & ABCA1 in macrophage, Both L-4F & the combination group improved the expressions of ABCA1, SR-BI & ABCG1 in liver & ABCA1 & ABCG1 in macrophage. 1P < 0.05, 2P < 0.001, vs. AS group; aP < 0.05, bP < 0.001, vs. Simva group;*P < 0.05, **P < 0.001, vs. L-4F group. Image collected & cropped by CiteAb from the following publication (//lipidworld.biomedcentral.com/articles/10.1186/1476-511X-12-180), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

ABCA1, ABCG1 & SR-BI protein expressions. a & b Hepatic protein expressions of ABCA1, ABCG1 & SR-BI were significantly decreased in COMT−/− mice at GD 18.5, compared to C57BL/6 J mice. Decreased hepatic ABCA1 expression was also observed at 10 days postpartum. ATI-5261 increased ABCA1 & ABCG1 expression in the liver at 10 days postpartum. c Placental protein expressions of ABCA1 & ABCG1 was reduced in COMT−/− mice, compared to C57BL/6 J mice. ATI-5261 treatment significantly increased ABCA1 levels in the placenta of COMT−/− mice. d Representative immunoblots of the corresponding proteins in the placenta with mouse RAW264.7 cell lysate included as positive control. Similar results were obtained when the experiment was repeated with lysates prepared from three batches of tissues. Data are presented as mean ± SEM. Groups (n = 8 in all groups) were compared using one-way ANOVA with post-hoc analysis (Tukey’s procedure). *, p < 0.05 Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30237900), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Effects of 13-HODE & LA in the presence & absence of PPAR alpha & PPAR gamma selective antagonists on molecular markers of cholesterol homeostasis in RAW264.7 macrophages. RAW264.7 cells were pre-treated without or with the PPAR alpha selective antagonist GW6471 or the PPAR gamma selective antagonist GW9662 & subsequently treated without (vehicle control) or with 2.5 μmol/L 13-HODE or 100 μmol/L LA for 24 h. Afterwards, cells were lysed & subsequently processed for western blotting as described in the materials & methods section. A, Representative immunoblots specific for ABCA1, ABCG1, SR-BI, LXR alpha , & beta -actin which was used for normalization are shown. B, Bars represent data from densitometric analysis & are means ± SD from three independent experiments (n = 3). Data are expressed as percentage of protein concentration of vehicle control cells. Results from statistical analysis are indicated: Significant effects are denoted with superscript letters. Bars marked without a common superscript letter significantly differ (P < 0.05). Image collected & cropped by CiteAb from the following publication (//lipidworld.biomedcentral.com/articles/10.1186/1476-511X-10-222), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - (A) Upregulation of ABCA1 & ABCG1 & (B) enhancement of cholesterol efflux by purple Perilla frutescens extracts (PPE) in 50 μg/ml Cu2+-oxidized low-density lipoproteins (LDL)-exposed J774A.1 murine macrophages. (A) For the measurement of ABCA1 & ABCG1 expression, total cell lysates were subjected to western blot analysis with a primary antibody against ABCA1 or ABCG1. beta -actin was used as an internal control. Bar graphs (means ± SEM, n=3) represent quantitative densitometric results of the upper bands. (B) Cholesterol efflux was expressed as the percentage of fluorescence in the medium relative to the total fluorescence. Bar graphs denoted without a common letter indicate significant difference, P<0.05. Image collected & cropped by CiteAb from the following publication (//www.spandidos-publications.com/10.3892/ijmm.2015.2101), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - 6‐Dihydroparadol increases A) ABCA1 & B) ABCG1, but not C) SR‐BI protein levels in cholesterol‐loaded THP‐1‐derived macrophages. THP‐1 cells were differentiated as described in Figure 2, & then loaded with unlabeled cholesterol for 24 h. Cells were treated with increasing concentrations of 6‐dihydroparadol (1–30 μm) for another 24 h. The protein levels of ABCA1, ABCG1, & SR‐B1 were detected by Western blot analysis. The control was treated with solvent vehicle (0.1% DMSO). As a positive control, TO901317 (5 μm, 24 h) was used. The bar graphs represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, & ***p < 0.001 versus control (determined by Student's t‐test or ANOVA with Bonferroni's post hoc test). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29802792), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Akt inhibition suppresses mTORC1 activity.BHK-ABCA1 cells were induced overnight with mifepristone (10 nM) & then incubated with DEBC, LY294002 or rapamycin at indicated concentrations for 2 h. Cells were then lysed & analyzed for ABCA1 & phosphorylated S6K by Western blotting. Actin was also blotted as loading control. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0113789), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Reversal of hepatic steatosis by reduction of SREBP-1 levels.(a) Western blotting analysis of SREBP-1 & ABCA1 levels in livers of miR-33+/+Srebf1+/+, miR-33+/+Srebf1+/−, miR-33−/−Srebf1+/+ & miR-33−/−Srebf1+/− mice. Representative western blot images are shown (n=4). Image collected & cropped by CiteAb from following publication (//pubmed.ncbi.nlm.nih.gov/24300912), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - 6‐Dihydroparadol increases A) ABCA1 & B) ABCG1 protein stability. THP‐1 cells were differentiated as described in Figure 2. Then cells were loaded with unlabeled cholesterol & treated with 6‐dihydroparadol (10 μm), or solvent vehicle (0.1% DMSO, control) for 24 h. Cells were lysed at different time points (0, 1, 2, 3, 4, & 6 h) after treatment with the protein synthesis inhibitor cycloheximide (CHX, 100 μm). The protein levels of both ABCA1 & ABCG1 were detected by Western blot analysis. The data points represent mean ± SD from three independent experiments. *p < 0.05 & **p < 0.01 versus control at the same time point (determined by Student's t‐test). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29802792), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Ligand activation of LXR inhibits LPS-induced MAP kinase activation through Abca1 induction.(A–D) Bone marrow-derived macrophages from Lxr alpha −/−Lxr beta −/− & control wild-type mice (A, B), or bone marrow-derived macrophages from myeloid-specific Abca1−/− & control wild-type mice (C, D) were pretreated with GW3965 (1 µM) overnight, followed by stimulation with LPS (10 ng/ml) for 20 min or 1 hr. Whole cell lysates were harvested & protein expression was analyzed by immunoblotting with the indicated antibodies (A, C). Protein expression was quantified by Image Quant TL7.0 (B, D). N = 4–6 per group. *p < 0.05, **p < 0.01, NS, not significant. Error bars represent means ± SEM.DOI://dx.doi.org/10.7554/eLife.08009.012 Image collected & cropped by CiteAb from the following publication (//elifesciences.org/articles/08009), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Western Blotting for ABCA1, as detailed in Supplementary Protocol #5 (Additional file 1). Caco-2 cells cultivated on filter inserts were treated for 48 h with bexarotene (RXR agonist, 5 μM), T0901317 (LXR agonist, 10 μM), GW3965 (LXR agonist, 10 μM) or the vehicle control (DMSO 0.1%). Protein expression was then determined by western blot analysis. Results were normalized for interexperimental variance & then set in relation to the vehicle control. Bar graphs represent mean ± SD from three independent experiments. A representative blot is shown - the bands stem from the same membrane & the vertical line indicates excised irrelevant bands. ns not significant, *P < 0.05, **P < 0.01 versus solvent vehicle (determined by paired t-test) Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/32308567), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Expression of ABCA1 & CROT in livers & RIP140 in macrophages is elevated in miR-33−/−Apoe−/− mice compared with miR-33+/+Apoe−/− mice. A, Quantitative real-time PCR analysis of Abca1, Crot, Cpt1a, & Prkaa1in livers from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. Values from miR-33+/+Apoe−/− mice were set at 100%. Values are mean±SE (n=9 to 11each); **P<0.01. B, Western analysis of ABCA1, CROT, CPT1a, & AMPK alpha in livers from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. beta -actin was used as a loading control. C, Densitometry of ABCA1, CROT, CPT1a, & AMPK alpha in livers from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. Values from miR-33+/+Apoe−/− mice were set at 100%. Values are mean±SE (n=4 each); *P<0.05. D, Total cholesterol, free cholesterol, cholesterol ester, & triglyceride levels in livers of miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. Values are mean±SE (n=9 to 11 each). E, HE staining of livers of miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice at age 20 weeks fed NC. Scale bar: 100 μm. F, Quantitative real-time PCR analysis of Nrip1 (RIP140) in peritoneal macrophages from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. Values from miR-33+/+Apoe−/− mice were set at 100%. Values are mean±SE (n=7 each). G, Western analysis of NRIP1 (RIP140) in peritoneal macrophages from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. GAPDH was used as a loading control. H, Densitometry of NRIP1 (RIP140) in peritoneal macrophages from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. Values from miR-33+/+Apoe−/− mice were set at 100%. Values are mean±SE (n=4 each); *P<0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23316322), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Basal ABCA1 expression & function in patient-derived iRPEs. (A) Representation of patient-derived iPSC lines & genotypes harboring polymorphisms in ABCA1 that are associated with decreased or increased risk for AMD development. (B) Relative expression of ABCA1 mRNA in patient-derived iRPEs under basal conditions normalized to RPL28 & decreased risk group. (C) Western blot analysis of ABCA1 levels in patient-derived iRPEs under basal conditions. Actin was detected as loading control. ABCA1 expression was quantified & normalized to actin. (D,E) Relative expression of ABCG1 (D) & NR1H3 (E) mRNA in patient-derived iRPEs under basal conditions normalized to RPL28 & decreased risk group. (F) Cholesterol efflux in patient-derived iRPEs after direct cell labeling & in the presence of ApoAI. (G) Cholesterol efflux in patient-derived iRPEs after phagocytosis of BODIPY-cholesterol-loaded POSs & in the presence of ApoAI. (H) Quantification of phagocytosed POSs per nuclei in patient-derived iRPEs. Values shown are means ± SD (n = 3). * p < 0.05; *** p < 0.001. Unpaired Student’s t-test. (I) Representative fluorescence microscopy images of phagocytosed FITC-labeled POSs (green) & staining for ZO-1 (red) & DAPI (blue) of increased risk cell line IPS19-00096. Scale bar = 100 μm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35328615), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - A–C) Effects of 6‐dihydroparadol & specific proteasomal, lysosomal, & calpain inhibitors on the ABCA1 protein levels. THP‐1 cells were differentiated as described in Figure 2, & then loaded with unlabeled cholesterol for 24 h. Cells were treated with or without 6‐dihydroparadol (6‐DP, 30 μm) for 24 h & incubated for another 3 h with or without the proteasome inhibitor lactacystin (10 μm), the lysosomal inhibitor chloroquine (100 μm), or the calpain inhibitor calpeptin (30 μg mL−1). The ABCA1 protein levels were detected by Western blot analysis. The bar graphs represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, & ***p < 0.001 versus control; n.s., not significant versus control (determined by Student's t‐test or ANOVA with Bonferroni's post hoc test). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29802792), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Suppressive effects of Ucn1 on foam cell formation & related protein expression in human monocyte-derived macrophages.Human monocytes were incubated for 7 days with RPMI-1640 supplemented with 10% human serum & the indicated concentrations of Ucn1, followed by a 19 h-incubation with 50 µg/ml oxLDL in the presence of 0.1 mmol/l [3H]oleate. Intracellular CE accumulation was determined from the radioactivity of cholesterol-[3H]oleate. Otherwise, before the addition of oxLDL, cells were harvested & subjected to immunoblotting analyses for CD36, ACAT1, or ABCA1. beta -Actin served as a loading control. Data are expressed as means ± SEM from 4–6 independent experiments with monocytes from 4–6 different donors. Baseline (1 fold) = 8.4±2.2 nmol/mg cell protein. *P<0.05, †P<0.001 vs. 0 nmol/l of Ucn1. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0110866), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Effect of evodiamine on ABCA1 transcription & the degradation rate of ABCA1 protein. (a) Differentiated THP-1 macrophages were incubated with 10 μM evodiamine or 10 μM pioglitazone as positive control for 24 h. Total RNA was extracted & ABCA1 mRNA expression levels were quantified by qRT-PCR. (b) 293 T cells were transfected with a luciferase reporter construct driven by the human ABCA1 promoter as described in the Materials & Methods section. After transfection, cells were treated with 10 μM Evodiamine or 1 μM T0901317 as positive control for 24 h. (c) Differentiated THP-1 macrophages were incubated for 24 h with (black circle) or without (Veh; white circle) evodiamine (10 μM) & lysed after addition of cycloheximide (CHX; 140 μM) at different time points (0, 10, 20, 40 min). Western blot analysis shows the decline of ABCA1 protein level with cycloheximide in the presence & absence of evodiamine. All data are means ± S.D. (n = 3) vs. solvent vehicle control (DMSO), *p < 0.05, **p < 0.01, n.s. no significance (ANOVA/Bonferroni). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30038271), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - miR-33 deficiency improved cholesterol efflux in macrophages. A, Quantitative real-time PCR analysis of Abca1 & Abcg1 in macrophages from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. Values from miR-33+/+Apoe−/− were set at 100%. Values are mean±SE (n=7 each); ***P<0.001. B, Western blotting analysis of ABCA1 & ABCG1 in thioglycollate-elicited peritoneal macrophages from miR-33+/+Apoe−/− & miR-33−/−Apoe−/− mice. GAPDH was used as a loading control. C, Cholesterol efflux from thioglycollate-elicited peritoneal macrophages in the presence or absence of apoA-I (5 or 10 μg/mL). Values are mean±SE (n=6 each); **P<0.01. D, Cholesterol efflux from thioglycollate-elicited peritoneal macrophages in the presence or absence of HDL-C (50 or 100 μg/mL). Values are mean±SE (n=6 each); **P<0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23316322), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Srebf1 is a miR-33 target gene.(a) Relative changes in lipid metabolism-related genes in the livers of miR-33−/− mice compared with miR-33+/+ mice fed NC at 16 weeks of age. (n=5–8 each,*P<0.05 in Student’s t-test). (b) Conservation of miR-33 target regions in the 3′UTR of Srebf1. Underlined sequences are the potential binding site of miR-33 seed sequences. * indicates the conservation among spieces. (c) 3′UTR reporter assay used to verify the target. Luciferase reporter activity of human & mouse SREBP-1 gene 3′UTR constructs in 293T cells overexpressing miR-control (miR-Con) & miR-33 (n=4 each, *P<0.05 & ***P<0.001 in Student’s t-test). (d) miR-33 dose-dependent changes in luciferase reporter activity of mouse Srebf1 3′UTR construct in 293T cells. miR-Con & miR-146a is used as a negative control (n=4 each, *P<0.05 & ***P<0.001 in one-way analysis of valiance test). (e) Luciferase reporter activity of the WT or mutant Srebf1 3′UTR at the potential miR-33 binding site in 293T cells (n=4 each, **P<0.01 in Student’s t-test). (f,g) Luciferase reporter activity of SRE-promoter (f) or FAS-promoter (g) in 293T cells. 293T cells were co-transfected with mouse Srebf1 with the full-length 3′UTR or without the 3′UTR, along with expression plasmids for miR-negative control, or miR-33. Values are the mean±s.e. (n=4 each, **P<0.01 versus miR-Con. ***P<0.001 versus miR-Con in one-way analysis of valiance test). (h) Western blotting analysis of SREBP-1, ABCA1, & IRS-2 in miR-33 transduced HepG2 cells & primary hepatocytes & hepatocytes prepared from miR-33+/+ & miR-33−/− mice. Representative western blot images are shown (n=4). Values are the means±s.e.m. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/24300912), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - (A) Chemical structure, (B) cytotoxicity of alpha -asarone, (C) enhancement of cholesterol efflux by alpha -asarone, (D & E) upregulation of ABCA1 & ABCG1 by alpha -asarone & beta -asarone, & (F) elevation of retinoid X receptor (RXR) alpha transcription. J774A.1 murine macrophages were exposed to 50 μg/ml oxidized low-density lipoprotein (LDL) & treated with 1–10 μM purple Perilla frutescens extracts (PPE)-alpha -asarone & 5–10 μM beta -asarone. (B) MTT assay was performed for the measurement of alpha -asarone toxicity. Graph data represent 1 of 4 independent experiments with multiple estimations. Values are expressed as the percentage cell survival relative to the untreated control cells (cell viability, 100%). (C) Cholesterol efflux was expressed as the percentage fluorescence in the medium relative to total fluorescence. (D & E) For the measurement of ABCA1 & ABCG1 expression, total cell lysates were subjected to western blot analysis with a primary antibody against ABCG1 or ABCG1. beta -actin was used as an internal control. Bar graphs (means ± SEM, n=3) represent quantitative densitometric results of the upper bands. Bar graphs denoted without a common letter indicate significant difference, P<0.05. (F) RXR alpha mRNA expression was measured by RT-PCR. GAPDH was used as a housekeeping gene for the co-amplification with RXR alpha . Image collected & cropped by CiteAb from the following publication (//www.spandidos-publications.com/10.3892/ijmm.2015.2101), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Bilirubin significantly decreases expression of ATP‐binding cassette transporter A1 (ABCA1) protein in THP‐1 macrophages treated with LXR agonist (TO901317) to upregulate ABCA1 protein. THP‐1 cells were differentiated for 72 hours with 200 nmol/L phorbol‐12‐myristate‐13‐acetate & then loaded with unlabeled cholesterol for another 24 hours. Cells were treated with TO901317 (5 μmol/L) or solvent vehicle (0.1% dimethyl sulfoxide [DMSO]) for 24 hours. Afterward, cells were treated with bilirubin (17.1 μmol/L) or solvent vehicle (0.1% DMSO) for 16 hours. The protein levels of ABCA1 were detected by western blot analysis. The bar graphs present mean±SD from 3 independent experiments. ***P<0.001 vs control (determined by ANOVA with Bonferroni post hoc test). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28455345), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Induction of cholesterol efflux attenuates the enhancement in HIV-1infectivity in NPCD55 cells. (A) Normal & NPCD55 cells were treated with 5 μM TO-901317 at 24 h post-infection & cultured for 72 h in the presence of the compound. At 96 h post-infection the cells were harvested & ABCA1, Gag, & beta -actin expression was detected by Western blotting analysis. All samples were loaded on the same gel. (B) AmplexRed assay was performed to measure free cholesterol content of untreated (filled) & TO-901317-treated (slashed) cells. Cholesterol content was normalized to protein concentration. (C) AmplexRed assay was performed to measure virion-associated cholesterol from purified virus produced in untreated (filled) & TO-901317-treated (slashed) infected cells. Virion-associated cholesterol content was normalized to p24 concentration. (D) TZM-bl reporter assay was performed to measure virus infectivity when cholesterol efflux was induced via TO-901317 stimulation. The ρ values were calculated by performing student T-test (*denotes < 0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/22273177), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Effects of apabetalone on ABCA1 expression & carboplatin response in ovarian cancer cells. (A) Apabetalone treatment reduced expression of ABCA1 in OVCAR-5 & OVCAR-5 CBPR cell lines. Cells were treated for 72 h with control medium (DMSO) or apabetalone (80 µM). Protein extracts from OVCAR-5 (~30 µg) were electrophoresed & immunoblotted with rabbit polyclonal ABCA1 antibody (1/1000, NB400-105, Novus Biological) & beta -actin (1/2000, Abcam) was used as a loading control. (B) Quantification of Western blot. OVCAR-5 (C), CaOV3 (D), OVCAR-5 CBPR (E) & CaOV3 CBPR (F) cell survival following treatment with carboplatin (CBP) alone (0-200 µM, black line) & in combination with apabetalone (Apa, 80 µM, purple line). (G) Carboplatin IC50 for OVCAR-5 & OVCAR-5 CBPR cells ± apabetalone. Data are mean ± SEM from three independent experiments. *P < 0.05, Student’s t-test. (H) Carboplatin IC50 for CaOV3 & CaOV3 CBPR cells ± apabetalone. Data are mean ± SEM from three independent experiments. P < 0.05, Student’s t-test. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35582032), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Expression of ATP‐binding cassette transporter A1 (ABCA1) protein in THP‐1 macrophages treated with bilirubin & in peripheral blood mononuclear cells (PBMCs) from Gilbert syndrome (GS) patients. A, Bilirubin suppresses the expression of ABCA1 protein in THP‐1‐derived macrophages time‐dependently. THP‐1 cells were differentiated as described in Figure 1 & then loaded with unlabeled cholesterol for another 24 hours. Cells were treated with bilirubin (17.1 μmol/L) for 4, 8, 16, & 24 hours. The protein levels of ABCA1 were detected by western blotting. Control was treated with solvent vehicle (0.1% dimethyl sulfoxide). The bar graph presents mean±SD from 3 independent experiments. B, Expression of ABCA1 protein is decreased in PBMCs from participants with high bilirubin blood levels (GS) compared with healthy controls. The protein levels of ABCA1 were detected by western blotting. The bar graph presents mean±SEM (n=28 per group). *P<0.05, **P<0.01 & ***P<0.001 vs control. ns indicates not significant (determined by Student t test). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/28455345), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - (A) Time course response of ABCA1 induction by pioglitazone, & (B) enhancement of ABCA1 & (C) peroxisome proliferator-activated receptor gamma (PPAR gamma ) by purple Perilla frutescens extracts (PPE). J774A.1 murine macrophages were incubated with 10 μM pioglitazone & 50 μg/ml oxidized low-density lipoprotein (LDL) in the absence or presence of 1–10 μg/ml PPE. For the measurement of expression of (A & B) ABCA1 & (C) PPAR gamma , total cell lysates were subjected to western blot analysis with a primary antibody against ABCA1 or PPAR gamma . beta -actin was used as an internal control. Bar graphs (means ± SEM, n=3) represent quantitative densitometric results of the upper bands. Bar graphs denoted without a common letter indicate significant difference, P<0.05. Image collected & cropped by CiteAb from the following publication (//www.spandidos-publications.com/10.3892/ijmm.2015.2101), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - LXR agonist-stimulated ABCA1 expression & function in patient-derived iRPEs. Patient-derived iRPE lines were stimulated for 16 h with LXR agonist & are relative to unstimulated cells shown in Figure 2. (A) Expression of ABCA1 mRNA normalized to RPL28 & relative to unstimulated cells shown in Figure 2B. (B) Western blot analysis of ABCA1 protein levels. Actin was detected as loading control. ABCA1 expression was quantified & normalized to actin. (C) Cholesterol efflux assay after direct cell labeling & in the presence of ApoAI. (D) Cholesterol efflux assay after phagocytosis of BODIPY-cholesterol-loaded POSs & in the presence of ApoAI. Data are relative to unstimulated cells shown in Figure 2F,G, respectively. Data are presented as means ± SD (n = 3). Unpaired Student’s t-test. * p < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35328615), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Akt inhibition by LY294002 or Akt1/2 also enhances cholesterol efflux to apoA-I from ABCA1-expressing BHK cells.A) BHK cells were induced as in Fig. 1 & then incubated with LY294002 (200 µM) for 2 h. Cells were then lysed & analysed for ABCA1, phosphorylated Akt (p-Akt) & total Akt by Western-blotting. Hsp70 was also blotted as loading control. B & C) BHK cells were labeled with [3H] cholesterol & induced with 10 nM mifepristone overnight. Cholesterol efflux was measured after 2 h incubation with BSA (1 mg/ml) or BSA plus apoA-I (5 µg/ml). Some of cells were also incubated with indicated doses of LY294002 (B) or Akt1/2 (C), in addition to apoA-I, during 2 h efflux period. Results are presented as the average of triplicate wells with standard deviation & representative of more than three independent experiments. *** P<0.0001, **P<0.001 & *P<0.05 vs apoA-I only. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0113789), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Generation, differentiation & analysis of ABCA1-deficient iRPEs. (A) Immunofluorescence staining for ZO-1 (red) in 4-week cultured iRPEs. Nuclei were stained with DAPI (blue). Scale bar = 100 μm. (B) Bright-field microscopy of iRPEs. (C) Ct values of RPE marker genes BEST1, OTX2, RLBP1 & RPE65 in iRPEs & parental iPSCs obtained by qPCR. Values shown are means ± SD (n = 3). Unpaired Student’s t-test. **** p < 0.0001. (D) Sequence alignment of parts of exon 14 (top) & exon 46 (bottom) of ABCA1-deficient iRPE cell clones & parental line. (E) Western blot of ABCA1 protein levels in ABCA1-deficient iRPE cell lines & parental cell line after 16 h of stimulation with 1 μM LXR agonist. Actin was detected as loading control. (F) Relative expression of ABCA1 mRNA in ABCA1-deficient iRPE cell lines & parental cell line normalized to ACTB. Values shown are means ± SD (n = 3). One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001. (G) Relative expression of ABCG1 mRNA in ABCA1-deficient iRPE cell lines & parental cell line normalized to ACTB. Values shown are means ± SD (n = 3). One-way ANOVA with Tukey’s post hoc test. ** p < 0.01; *** p < 0.001. (H) Cholesterol efflux assay in iRPEs in the presence of ApoAI and/or LXR agonist (LXR ag). Values shown are means ± SD (n = 3). Two-way ANOVA with Bonferroni post hoc test vs. DMSO + BSA control. **** p < 0.0001. (I) Bright-field (BF) microscopy & Nile Red fluorescence microscopy (overview & close-up view marked with white squares) of 4-week cultured ABCA1-deficient iRPE cell lines & parental cell line. Relative fluorescence was quantified & is shown as mean ± SD (n = 8). One-way ANOVA with Tukey’s post hoc test. * p < 0.05, **** p < 0.0001. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35328615), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Akt inhibition enhances cholesterol efflux to apoA-I from wt MEFs, but not from TSC2-/- MEFs where mTORC1 is constitutively activated.A) Mouse embryonic fibroblasts (MEFs), wt & TSC2-/-, were induced overnight with T0901317 (10 µM). Cell lysates were collected for Western blotting for phosphorylated S6K & total S6K. B) wt & TSC2-/- MEFs were incubated with or without T0901317 (10 µM) overnight. Some of cells were then incubated with apoA-I (10 µg/ml) or apoA-I plus DEBC (25 µM) for 2 h. The expression of ABCA1, phosphorylated Akt, total Akt & loading control hsp70 were analyzed by Western blotting. C) wt & TSC2-/- were labeled with [3H] cholesterol for 1 day & then incubated with or without T0901317 (10 µM) overnight. Some of cells were then incubated with apoA-I (10 µg/ml) or apoA-I plus DEBC (25 µM) for 2 h to analyze cholesterol efflux. Data presented as the average of triplicate wells with standard deviation & representative of at least three independent experiments. *** P<0.0001 vs apoA-I only. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0113789), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Akt inhibition by DEBC enhances cholesterol efflux to apoA-I specifically from ABCA1-expressing BHK cells.A) BHK-ABCA1 cells were induced with mifepristone (10 nM) overnight & then incubated with indicated doses of DEBC for 2 h. Cells were then lysed & Western-blotted for ABCA1, phosphorylated Akt (p-Akt) & total Akt. Hsp70 was also blotted as loading control. B) BHK-ABCA1 cells were labeled with [3H] cholesterol & induced overnight as above. After 2 h incubation with BSA (1 mg/ml) or BSA plus apoA-I (5 µg/ml), cholesterol efflux was measured as described in the Methods section. Some of the cells were also incubated with indicated doses of DEBC, in addition to apoA-I, during 2 h efflux period. C) BHK-ABCA1 & BHK-A937V cells were induced with mifepristone (10 nM) overnight. 2 h Cholesterol efflux was measured as above either with or without DEBC (25 µM). Results are presented as the average of triplicate wells with standard deviation & representative of more than three independent experiments. *** P<0.0001 & **P<0.001 vs apoA-I only. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0113789), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - LXRs control expression of genes involved in cholesterol homeostasis & fatty acid synthesis in MPECs.(A) qPCR analysis of Abca1, Abcg1 & Idol levels in WT (+/+) & Lxr alpha beta −/− (lxr−/−) MPECs after DMSO (vehicle) or T0901317 stimulation (B) Effect of 9-cis retinoic acid and/or T0901317 stimulation on Abca1 accumulation levels in WT (+/+) & Lxr alpha beta −/− (lxr−/−) MPECs (C) qPCR analysis of Srebp1, Acc, & Fas levels in WT (+/+) & Lxr alpha beta −/− (lxr−/−) MPECs after DMSO (vehicle) or T0901317 stimulation. (qPCR analysis results from 4 independant experiments & was normalized using 36b4 gene). *p<0.05, **p<0.01, ***p<0.001 in Student’s t test. Error bars represent mean ± SEM. (D) Western blot analysis was performed on WT (+/+) or Lxr alpha beta −/− MPECs using Srebp1c, Abca1 & beta -Actin antibodies. (E) Oil-Red O staining (ORO) & Normarski/Dapi of WT (+/+) & Lxr alpha beta −/− MPECs, or WT MEFs, treated for 48h with DMSO (vehicle) or T0901317 (1 µM). Head arrows indicate lipid droplets. Scale bars 100 µm. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23554947), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Anti-ApoA-1 IgGs upregulate ABCA1. ABCA1 was increased after 16 & 24 h of anti-ApoA-1 IgG exposure to HMDM at the mRNA level as revealed by RT-PCR (a) as well as at the protein level after 24 h anti-apoA-1 IgG stimulation, as revealed by Western blot analysis (b). In panel a, data are expressed as fold change expression of the mean ± SD of ABCA1 calculated by delta delta CT method of six independent experiments (n = 6) & values were normalized to untreated condition. p-values were calculated using the Student’s t-test: * p < 0.05, ** p < 0.01, **** p < 0.0001. (b) One of four representative Western blots is shown. Data are the mean ± SD of band intensity volume/actin intensity volume of four independent experiments (n = 4). p-values were calculated using the Student’s t-test: * p = 0.02, ** p = 0.005. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31766415), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - ABCA1 protein expression is upregulated in ovarian cancer cells with acquired carboplatin resistance. ABCA1 expression in OVCAR-5 (A) & CaOV3 (C) & carboplatin resistant OVCAR-5 CBPR (B) & CaOV3 CBPR (D) by immunocytochemistry using rabbit polyclonal ABCA1 antibody (1/100, NB400-105, Novus Biological). (E) OVCAR-5 cells with Rabbit IgG & (F) CaOV3 cells with Rabbit IgG. (G) Protein extracts from OVCAR-5 (~30 µg) & CaOV3 cell lines (~60 µg) were electrophoresed & immunoblotted with rabbit polyclonal ABCA1 antibody (1/1000, NB400-105, Novus Biological), & beta -actin (1/2000, Abcam) was used as a loading control. A major band was detected at ~250 kDa, which is the predicted size for ABCA1. (H) Quantitation of ABCA1 Western blots. Data are from 2-4 independent experiments. Statistical significance was determined using the Student’s t-test, *P < 0.05. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/35582032), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Evodiamine enhances apo A1-mediated ChE from THP-1 macrophages & increases ABCA1 protein level. (a) Differentiated THP-1 cells were loaded with [3H]-cholesterol together with the indicated treatments for 24 h. On the next day, the cells were washed twice with PBS & incubated with the same compounds [solvent vehicle control (Veh; ≤0.1% DMSO), evodiamine (1–20 μM), & the PPAR gamma agonist pioglitazone (10 μM) as positive control] with or without 10 µg/mL apo A1. Extracellular as well as intracellular radioactivities were quantified with scintillation counter. Differentiated THP-1-derived macrophages were treated with solvent vehicle control (Veh; ≤0.1% DMSO), evodiamine (10 μM), & the PPAR gamma agonist pioglitazone (10 μM) as positive control. After 24 h incubation, the cells were lysed & 20 μg protein was resolved via SDS-PAGE. Immunodetection was performed with antibodies against the indicated proteins, ABCA1 (b), ABCG1 (c), & SR-B1 (d), & visualized by chemiluminescence detection. All experiments were performed at least three times & data are presented as means ± S.D. vs. solvent vehicle control, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance (ANOVA/Bonferroni). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/30038271), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Functional miR-33 responsive elements in the 3′UTR of ATP8B1 & ABCB11A,B. Conserved sequences in the 3′UTR of ATP8B1 & ABCB11 are partially complementary to miR-33. The element in human ATP8B1 is located 1877–1897 nt after the stop codon. The element in ABCB11 overlaps the stop codon in primates, while rodents show a conserved sequence 732–751 nt after the stop codon.C,D. Luciferase assays in HEK293 cells using the whole 3′UTR of human or murine ATP8B1 & ABCB11, or a single copy of the responsive elements (RE) identified above, or mutant responsive elements (RE*; where AATGCA was mutated to GGGTTG to prevent complementarity to the seed sequence of the miRNA), co-transfected with (closed bars) or without (open bars) a vector to overexpress miR-33. In grey, data from empty (negative control) & R33 (positive control containing a 100% match to miR-33) reporter vectors.E,F. Relative mRNA expression of canalicular transporters in primary murine hepatocytes (n = 4 dishes/condition) & human HuH-7 hepatoma cells (n = 3 dishes/condition) transduced 48 h with empty or miR-33 adenovirus.G. Relative protein levels in HuH7 cells transduced with empty or miR-33 adenovirus. Some cells were incubated for 16 h in the presence of FXR:RXR agonists (2 µmol/L GW4064 : 1 µmol/L 9-cis-retinoic acid) to induce ABCB11. Asterisk indicates a non-specific band. Data shown as mean ± SD; **p < 0.01. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/22767443), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - Gas6 treatment enhances expression of LXR alpha & LXR beta & their target genes in BMDM.Mouse BMDM were stimulated with 400 ng/ml Gas6, 1 μM T0901317, 10 ng/ml interferon (IFN)-gamma , 10 ng/ml IL-4, or 100 ng/ml LPS for 4 h (a,b) or 400 ng/ml Gas6 for the indicated times (c–g). (a,b,e) The amounts of the LXR alpha , LXR beta , ABCA1, ABCG1, ApoE, AIM, Arg2, VEGF, YM1, & Arg1 mRNAs were analyzed by real-time PCR & normalized to that of Hprt mRNA. (c,d,f,g) The relative abundances of LXR alpha , LXR beta , ABCA1, ABCG1, ApoE, Aim, Arg2, & VEGF proteins were determined by Western blotting analysis. The relative densitometric intensity was determined for each band & normalized to beta -actin. Data in all bar graphs are means ± SEM of three independent experiments. *P < 0.05 compared with control. Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27406916), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: ABCA1 Antibody - BSA Free [NB400-105] - (A) Time course response of ABCA1 induction by TO-091317 & (B) upregulation of ABCA1 by purple Perilla frutescens extracts (PPE), & (C) enhancement of liver X receptor (LXR) alpha induction by PPE. J774A.1 murine macrophages were cultured with 1 μM TO-091317 or 50 μg/ml Cu2+-oxidized low-density lipoprotein (LDL) in the absence or presence of 1–10 μg/ml PPE. For the measurement of expression of (A & B) ABCA1 & (C) LXR alpha , total cell lysates were subjected to western blot analysis with a primary antibody against ABCA1 or LXR alpha . beta -actin was used as an internal control. Bar graphs (means ± SEM, n=3) represent quantitative densitometric results of the upper bands. Bar graphs denoted without a common letter indicate significant difference, P<0.05. Image collected & cropped by CiteAb from the following publication (//www.spandidos-publications.com/10.3892/ijmm.2015.2101), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Reactivity:

Hu, Mu, Rt, Po, Ca, Ch, ChHa, Eq, Ha, Md, Pm, Rb

Species Glossary

Reactivity: Hu, Mu, Rt, Po, Ca, Ch, ChHa, Eq, Ha, Md, Pm, Rb

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	NB400-105AF350
Alexa Fluor 405	NB400-105AF405
Alexa Fluor 488	NB400-105AF488
Alexa Fluor 532	NB400-105AF532
Alexa Fluor 594	NB400-105AF594
Alexa Fluor 647	NB400-105AF647
Alexa Fluor 700	NB400-105AF700
Alexa Fluor 750	NB400-105AF750
Allophycocyanin	NB400-105APC
Allophycocyanin/Cy7	NB400-105APCCY7
Biotin	NB400-105B
CoraFluor 1	NB400-105CL1
DyLight 350	NB400-105UV
DyLight 405	NB400-105V
DyLight 488	NB400-105G
DyLight 550	NB400-105R
DyLight 594	NB400-105DL594
DyLight 650	NB400-105C
DyLight 680	NB400-105FR
DyLight 755	NB400-105IR
FITC	NB400-105F
HRP	NB400-105H
Janelia Fluor 525	NB400-105JF525
Janelia Fluor 549	NB400-105JF549
Janelia Fluor 646	NB400-105JF646
Janelia Fluor 669	NB400-105JF669
PE	NB400-105PE
PE/Atto594	NB400-105PEATT594
PE/Cy5.5	NB400-105PECY55
PE/Cy7	NB400-105PECY7
PerCP	NB400-105PCP
mFluor Violet 450 SE	NB400-105MFV450
mFluor Violet 500 SE	NB400-105MFV500
mFluor Violet 610 SE	NB400-105MFV610

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB400-105SS

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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

Category

Protein/Target

Applications

Reactivity

Citation

Antibody Validation

Reviews

Host

Available Conjugates

Format

Marker

Modification

Clonality

Clone

Gene Symbol

Images Type

Isotype

Kit Type

Lysate Life Stage

Lysate Subcellular Fraction

Lysate Tissue

Lysate Type

Preservative

Protein Or Peptide

Lysate Tissue Condition

Research Areas

Has Sample Size

Promotional Item

Species Glossary