Detection of Mouse IL‑1 beta/IL‑1F2 by Simple WesternTM.
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 (
HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
Cell Proliferation Induced by IL‑1 beta/IL‑1F2 and Neutralization by Mouse IL-1 beta/IL-1F2 Antibody.
Recombinant Mouse IL-1 beta/IL-1F2 (
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 ND
50 is typically ≤ 0.25 µg/mL.
Detection of IL-1 beta/IL-1F2 in Mouse Intestine.
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 (Catalog #
VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog #
VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in epithelial cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/28148962), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/30846986), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Western Blot
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 (https://dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/34000990), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by In vivo assay
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 (https://dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Western Blot
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, along with 200ng of plasmids encoding mCASP-1 and mIL-1 beta, and 200ng empty vector to normalize plasmid quantities. Lower quantities of NLRP1B were transfected as compared to panel D so to avoid spontaneous NLPR1B activation. For panels B-E, data shown are representative of at least three similar experiments. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/30846986), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/26059659), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
The ESX-1 secretion system of Mtb does not affect secretion of proinflammatory cytokines in dendritic cells but is important for complete inflammasome activation.BMDCs were left uninfected (UI), infected with wild-type Mtb (Mtb) or the esxA deletion mutant ( deltaesxA) for 4 h at MOI of 10, washed and incubated for an additional 24 h (A+B) or the indicated timepoints (C+D). (A) The cytokine profile in the supernatants was analyzed using a bead-based immunoassay (black = uninfected, white = Mtb, gray = deltaesxA). (B) and (C) IL-1 beta secretion was analyzed by ELISA. (D) The percent of cells with activated caspase-1 was determined via flow cytometry using fluorescent caspase-1 substrates (FLICA). (E) Pro-IL-1 beta protein levels in BMDCs. (F) GFP-labeled bacteria were used to infect BMDCs and rate of infection was determined via flow cytometry. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three. In all figures, the asterisks denote range of p values (* = p<0.05, ** = 0.01>p>0.001,***p<0.001, ns = not significant ) as determined by one way ANOVA with Tukey's post test. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0040722), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by In vivo assay
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 (https://dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by In vivo assay
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 (https://dx.plos.org/10.1371/journal.pone.0115827), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/35305649), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 alphaHis (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 (https://dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 alphaHis (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 (https://dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Western Blot
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, along with 200ng of plasmids encoding mCASP-1 and mIL-1 beta, and 200ng empty vector to normalize plasmid quantities. Lower quantities of NLRP1B were transfected as compared to panel D so to avoid spontaneous NLPR1B activation. For panels B-E, data shown are representative of at least three similar experiments. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://www.nature.com/articles/ncomms7115), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Western Blot
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 (https://dx.plos.org/10.1371/journal.ppat.1006052), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/30315153), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/28148962), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://dx.plos.org/10.1371/journal.ppat.1006126), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/31244838), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/28824884), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
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 (https://www.nature.com/articles/ncomms11929), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse Mouse IL-1 beta/IL-1F2 Antibody by Western Blot
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 (https://pubmed.ncbi.nlm.nih.gov/36522378), licensed under a CC-BY license. Not internally tested by R&D Systems.
