Product Search for il 2 r beta: Novus Biologicals

M6000B

Mouse IL-6 Quantikine ELISA Kit, 2 Plate

(717 Publications)

1 Kit (1 Plate)

M6000B

Increased production of proinflammatory cytokines and VEGF in the plasma and chondrocytes in high fat diet (HFD)-feeding mice.(a~d) levels of plasma IL-6 (a), TNF-alpha (b), IL-1 beta (c) and VEGF (d) in the C57BL/6 male mice fed with a normal diet (ND) or HFD for 8 weeks. (n = 5, **P < 0.01). (e~h) Relative mRNA expression of IL-6 (e), TNF-alpha (f), IL-1 beta (g) and VEGF (h) in the chondrocytes isolated from the 8-week ND or HFD-feeding mice. (n = 5, **P < 0.01). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/26271607), licensed under a CC-BY license. Not internally tested by R&D Systems.

PYC suppressed LPS-induced proinflammatory cytokine production in BV2 microglia.Cells were incubated with the indicated concentrations of PYC or vehicle for 1h before LPS treatment (500ng/mL). After 24 h incubation, the culture supernatants were collected, and the amount of TNF-alpha , IL-6, IL-1 beta and IL-10 were measured by ELISA (A-D). Data are represented as mean ± SD from at least 3 independent experiments. *P< 0.05 compared with LPS alone. #P< 0.05. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/26367267), licensed under a CC-BY license. Not internally tested by R&D Systems.

FFA and lactate enhance production of proinflammatory cytokines and VEGF via HIF1 alpha in mouse chondrocytes.(a) Immunoblotting assay of HIF1 alpha in primary chondrocytes transfected with a scramble siRNA (siNC, 20 nM), a mouse HIF1 alpha specific siRNA1 (siH-1, 20 nM) or siRNA2 (siH-2, 20 nM) for 36 h. (b~e) Relative mRNA levels of IL-6 (b), TNF-alpha (c), IL-1 beta (d) and VEGF (e) in the mouse primary chondrocytes transfected with siNC (20 nM), siH-1 (20 nM) or siH-2 (20 nM)for 12 h plus additional treatment with BSA (5%) or FFA (200 μM) for 24 h. Values not sharing a common superscript letter differ significantly. (n = 3, P < 0.05). (f~i) Relative mRNA levels of IL-6 (f), TNF-alpha (g), IL-1 beta (h) and VEGF (i) in the mouse primary chondrocytes transfected with siNC (20 nM), siH-1 (20 nM) or siH-2 (20 nM) for 12 h plus additional treatment with lactate (25 mM) or vehicle control for 24 h. Values not sharing a common superscript letter differ significantly. (n = 3, P < 0.05). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/26271607), licensed under a CC-BY license. Not internally tested by R&D Systems.

$HFD-induced plasma saturated free fatty acid (FFA) and lactate trigger production of proinflammatory cytokines and VEGF in mouse chondrocytes.(a~d) Relative mRNA levels of IL-6 (a), TNF-alpha (b), IL-1 beta (c) and VEGF (d) in the mouse primary chondrocytes treated with full or fractionated (>3 KD or <3 KD) serum from the 8-week ND or HFD-feeding mice. (n = 3, *P < 0.05, **P < 0.01 and ***P < 0.005). (e~h) Relative mRNA levels of IL-6 (e), TNF-alpha (f), IL-1 beta (g) and VEGF (h) in the mouse primary chondrocytes treated with BSA (5%) or BSA-linked FFA (18:0, 200 μM) for 24 h. (n = 3, *P < 0.05 and **P < 0.01). (i~l) Relative mRNA expression of IL-6 (i), TNF-alpha (j), IL-1 beta (k) and VEGF (l) in the mouse primary chondrocytes treated with lactate (25 mM) or vehicle control for 24 h. (n = 3, *P < 0.05 and **P < 0.01). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/26271607), licensed under a CC-BY license. Not internally tested by R&D Systems.$

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

Applications:

Conjugate	Catalog #	Availability	Size	Price
HRP	PM6000B
HRP	SM6000B

compare

(select up to 3 products from this page to compare)

201-LB

Recombinant Human IL-1 beta/IL-1F2 Protein

(643 Publications)

5 μg

201-LB

1 μg/lane of Recombinant Human IL-1 beta /IL-1F2 was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing a single band at 17 kDa.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	201-LB-005/CF
Carrier Free	201-LB-025/CF
Carrier Free	201-LB-500/CF
Carrier Free	201-LB-100/CF
Carrier Free	201-LB-010/CF
Carrier Free	201-LB-01M/CF

compare

(select up to 3 products from this page to compare)

DY406

Mouse IL-6 DuoSet ELISA, 15 Plate

(691 Publications)

1 Kit (for 5 Plates)

DY406

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

Applications:

compare

(select up to 3 products from this page to compare)

AF-401-NA

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

(3 Reviews)

(610 Publications)

100 μg

AF-401-NA

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

, , , ,

Applications: , , , ,

Host:

Goat Polyclonal

Host: Goat Polyclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

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

Available Formulations

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

compare

(select up to 3 products from this page to compare)

DY206

Human IL-6 DuoSet ELISA, 15 Plate

(657 Publications)

1 Kit (for 5 Plates)

DY206

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

compare

(select up to 3 products from this page to compare)

DY401

Mouse IL-1 beta/IL-1F2 DuoSet ELISA, 15 Plate

(576 Publications)

1 Kit (for 5 Plates)

DY401

Exosomal release of IL-1 alpha by neutrophils(A) Representative confocal images of peritoneal neutrophils stimulated with LPS or curdlan for 6 h.(B) Quantification of IL-1 alpha and CD63 co-localization using ImageJ (each data point represents a single cell).(C) NTA of EV size distribution and concentration.(D–G) Neutrophils were stimulated in the presence of exosome inhibitor GW4869, and IL-1 alpha and IL-1 beta were quantified by ELISA in isolated EVs following lysis (D and F) and in total cell-free supernatants (E and G).(H) Inhibition of EV secretion shown by NTA.(I) Bioactive IL-1 signaling through IL-1R1 reporter cells was measured in isolated exosomes in the absence of detergent lysis (n = 4). Neutralizing antibodies (Abs) to IL-1 alpha , IL-1 beta , or both cytokines were included in the reporter assay, and bioactive cytokine concentration was calculated based on a standard curve using recombinant IL-1 alpha and IL-1 beta .Two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Experiments in (A) and (B) were repeated three times; (C)–(G) are biological replicates from repeat experiments. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34010648), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

Applications:

compare

(select up to 3 products from this page to compare)

MLB00C

Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit, 2 Plate

(559 Publications)

1 Kit (1 Plate)

MLB00C

Reactivity:

Mu

Species Glossary

Reactivity: Mu

Applications:

Applications:

Conjugate	Catalog #	Availability	Size	Price
HRP	SMLB00C
HRP	PMLB00C

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	SMLB00C
Carrier Free	PMLB00C

compare

(select up to 3 products from this page to compare)

NB600-501

beta-Actin Antibody (AC-15)

(27 Reviews)

(644 Publications)

0.1 ml

NB600-501

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Reactivity:

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

Species Glossary

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

Applications:

, , , , , , , , , , ,

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

Host:

Mouse Monoclonal

Host: Mouse Monoclonal

Sizes:

Sample Size Available

Sizes: Sample Size Available

Conjugate:

Unconjugated

Available Conjugates:

Validated by:

Validated by:

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

Available Formulations

Formulation	Catalog #	Availability	Price
BSA Free	NB110-67828

compare

(select up to 3 products from this page to compare)

HS600C

Human IL-6 Quantikine HS ELISA Kit

(527 Publications)

1 Kit

HS600C

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Conjugate	Catalog #	Availability	Size	Price
HRP	SS600C
HRP	PHS600C

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	SS600C
Carrier Free	PHS600C

compare

(select up to 3 products from this page to compare)

DY201

Human IL-1 beta/IL-1F2 DuoSet ELISA, 15 Plate

(422 Publications)

1 Kit (for 5 Plates)

DY201

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

compare

(select up to 3 products from this page to compare)

7270-IL

Recombinant Human IL-6 (HEK293-expressed) Protein

(11 Publications)

25 μg

7270-IL

Measured in a cell proliferation assay using T1165.85.2.1 mouse plasmacytoma cells. The ED<sub>50</sub> for this effect is 0.1-0.6 ng/mL.

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Available Formulations

Formulation	Catalog #	Availability	Price
Carrier Free	7270-IL-025/CF
Carrier Free	7270-IL-010/CF
Carrier Free	7270-IL-100/CF

compare

(select up to 3 products from this page to compare)

DLB50

Human IL-1 beta/IL-1F2 Quantikine ELISA Kit

(362 Publications)

1 Kit

DLB50

Reactivity:

Hu

Species Glossary

Reactivity: Hu

Applications:

Applications:

Conjugate	Catalog #	Availability	Size	Price
HRP	PDLB50
HRP	SLB50

compare

(select up to 3 products from this page to compare)

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

Modification

Clonality

Clone

Gene Symbol

Images Type

Isotype

Preservative

Protein Or Peptide

Research Areas

Has Sample Size

Promotional Item

Species Glossary

Mouse IL-6 Quantikine ELISA Kit, 2 Plate

Recombinant Human IL-1 beta/IL-1F2 Protein

Mouse IL-6 DuoSet ELISA, 15 Plate

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

Human IL-6 DuoSet ELISA, 15 Plate

Mouse IL-1 beta/IL-1F2 DuoSet ELISA, 15 Plate

Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit, 2 Plate

beta-Actin Antibody (AC-15)

Human IL-6 Quantikine HS ELISA Kit

Human IL-1 beta/IL-1F2 DuoSet ELISA, 15 Plate

Recombinant Human IL-6 (HEK293-expressed) Protein

Human IL-1 beta/IL-1F2 Quantikine ELISA Kit