Cells and tissues were lysed by a RIPA buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS,150mM NaCl, and 25mM Tris-HCl, pH 7.6) with protease inhibitor cocktail (11836170001; Roche). The proteins of the supernatant for the IL-1β secretion inflammasome assay were precipitated by chloroform and methanol. These lysates were prepared by a sample buffer (NP0007; Invitrogen) with 10 % 2-Mercaptoethanol and loaded onto 4–20% precast polyacrylamide gels (4561096, 5671095; Bio-Rad). Imaging was performed with enhanced chemiluminescent detection (34096; Thermo Scientific) on an Azure Biosystems c600 imager. Antibodies; anti-H3K9bhb: PTM-1250, PTM Biolabs; anti-H3K9ac: #9496, Cell Signaling; anti-H3: 07–690, Millipore; anti-Gapdh: 60004–1-lg, Proteintech; anti-HSP90: 610419, BD Biosciences; anti-Kac: #9441, Cell Signaling; anti-Kbhb: PTM-1201, PTM-Biolabs; anti-IL-1β: GTX74034, GeneTex; anti-mouse IgG: #7076, Cell Signaling; anti-rabbit IgG: #7074, Cell Signaling.
Anti il 1β
Manufactured by GeneTex
Sourced in United States
Anti-IL-1β is a laboratory reagent that can be used to detect and quantify the cytokine interleukin-1 beta (IL-1β) in biological samples. It functions as a specific antibody that binds to IL-1β, allowing for its identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Proteintech (2 mentions)
Anti caspase 1 p20, by Adipogen (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
C600 imager, by Azure Biosystems (1 mentions)
Np0007, by Thermo Fisher Scientific (1 mentions)
Anti h3, by Merck Group (1 mentions)
Anti hsp90, by BD (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Enhanced chemiluminescent detection, by Thermo Fisher Scientific (1 mentions)
Anti h3k9ac, by Cell Signaling Technology (1 mentions)
Ptm 1201, by PTM Biolabs (1 mentions)
Clarity max western ecl substrate, by Bio-Rad (1 mentions)
Anti cith3, by Abcam (1 mentions)
Anti asc, by GeneTex (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Anti caspase 1, by GeneTex (1 mentions)
Anti nlrp3, by GeneTex (1 mentions)
Odyssey classic imager, by LI COR (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Anti gsdmd, by Abcam (1 mentions)
10 protocols using anti il 1β
1
Immunoblotting for Inflammasome Activation
2
Detecting Pro-Caspase-1 and IL-1β by Western Blot
3
Western Blot Analysis of Inflammatory Markers
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Cell lysates and supernatants were routinely processed as described previously. Additionally, cell lysates were prepared according to a previous detailed description39 (link) for ASC oligomerization.
Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore; MA, USA) after electrophoresis. The membranes were blocked with 8% skimmed milk for 1 h at RT and incubated with primary antibodies, including anti-cit-H3 (1:1000, Abcam, MA, USA), anti-NE (1:1000, R&D Systems, MN, USA), anti-cleaved caspase-1 (1:1000, GeneTex; CA, USA), anti-IL-1β (1:1000, GeneTex; CA, USA), anti-ASC (1:1000, GeneTex; CA, USA) and anti-GAPDH (1:1000, Proteintech, Wuhan, China) antibodies, overnight at 4°C. After three washes (Tris-buffered saline plus 0.05% Tween-20), the membranes were incubated with a horseradish peroxidase-conjugated goat-anti rabbit or mouse secondary antibody (SAB; MD, USA) for 1 h at RT on a shaker. Signals were detected with ClarityMax Western ECL Substrate (Bio-Rad, CA, USA) and quantified by ImageJ software (Rawak Software Inc., Germany).
Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore; MA, USA) after electrophoresis. The membranes were blocked with 8% skimmed milk for 1 h at RT and incubated with primary antibodies, including anti-cit-H3 (1:1000, Abcam, MA, USA), anti-NE (1:1000, R&D Systems, MN, USA), anti-cleaved caspase-1 (1:1000, GeneTex; CA, USA), anti-IL-1β (1:1000, GeneTex; CA, USA), anti-ASC (1:1000, GeneTex; CA, USA) and anti-GAPDH (1:1000, Proteintech, Wuhan, China) antibodies, overnight at 4°C. After three washes (Tris-buffered saline plus 0.05% Tween-20), the membranes were incubated with a horseradish peroxidase-conjugated goat-anti rabbit or mouse secondary antibody (SAB; MD, USA) for 1 h at RT on a shaker. Signals were detected with ClarityMax Western ECL Substrate (Bio-Rad, CA, USA) and quantified by ImageJ software (Rawak Software Inc., Germany).
4
Western Blot Analysis of Colon Tissue Proteins
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Proteins were extracted from colon tissue samples using RIPA lysis buffer (Solarbio life sciences, Beijing, China). After denaturation, the protein extracts were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA) for 2 h. PVDF membranes were probed overnight at 4 ℃ using the corresponding primary antibodies: anti- GPR43, anti-NLRP3, anti-Caspase-1, anti-IL-8 and anti-IL-1β (GeneTex, CA, USA). After washing thrice with Tris-buffered saline, the membranes were incubated at 25 ℃ for 1 h with the HRP-conjugated secondary antibody. Antibody binding was determined using a chemiluminescence reagent. The membrane was then scanned and analyzed using a Bio-Rad analyzer (Hercules, CA, USA). The relative amount of target protein was calculated using the gray value ratios of the protein to GAPDH.
5
Immunoblot detection of caspase-1 and tissue factor
6
Immunoblot Analysis of Inflammasome Markers
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For detection of Caspase-1, IL-1β, GSDMD, and NINJ1 by immunoblot, cells were washed with cold PBS and lysed with LDS sample buffer. Culture supernatant was precipitated with 1/10 volume of 2% sodium cholate and 1/10 volume of 100% trichloroacetic acid (TCA), and then dissolved in LDS sample buffer. TF was detected using anti-tissue factor (R&D, Cat#AF3178-SP) at 1:1000 dilution. Caspase-1 was detected using anti-Caspase-1 (Adipogen, Cat#AG-20B-0042-C100) at 1:1000 dilution. IL-1β was detected using anti-IL-1β (GeneTex, Cat#GTX74034) at 1:1000 dilution. GSDMD was detected using anti-GSDMD (Abcam, Cat#ab219800) at 1:1000 dilution. NINJ1 was detected using anti-NINJ1 (kindly provided by Genentech, Cat#Ninj1-rbIgG-25:10363). Tissue fibrin was detected using anti-fibrin (59D8) at 1 mg/ml. Images were acquired with LI-COR Odyssey Imager.
7
Western Blot Analysis of Inflammatory Signaling
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Proteins in the lysate were separated on a denaturing polyacrylamide gel and transferred to a polyvinylidene fluoride (polyvinylidene difluoride) membrane (Merck Millipore, Burlington, MA). The membrane was incubated with 5% skim milk (BD Biosciences) in Tris-buffered saline with 0.1% Tween 20 (TBST) buffer and the primary antibodies, namely, anti-IL-1β (GTX130021; GeneTex, Irvine, CA), anti-GSDMDC1 (sc-81868; Santa Cruz Biotechnology), anti-β-actin (sc-47778; Santa Cruz Biotechnology), anti-caspase-1 (3866S; Cell Signaling Technology), anti-pan-flavivirus E (4G2, purified in the lab), anti-C3 (ab200999; Abcam), anti-C/EBP-β (90081S; Cell Signaling Technology), anti-C/EBP-β (LAP) (3087S; Cell Signaling Technology), anti-phospho-C/EBP-β (Thr235) (3084S; Cell Signaling Technology), anti-p38 MAPK (9212S; Cell Signaling Technology), anti-phospho-p38 MAPK (9211S; Cell Signaling Technology), anti-p44/42 MAPK (9102S; Cell Signaling Technology), and anti-phospho-p44/42 MAPK (4370S; Cell Signaling Technology). Horseradish peroxidase-conjugated secondary antibodies from Bio-Rad and enhanced chemiluminescence reagents (Thermo Fisher Scientific) were used for protein detection.
8
Immunoblotting of Cell Death Pathway
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Antibodies against caspase-1, NLRP3 and ASC were made in house and were described previously14 (link)25 (link)51 (link). Anti-DRP1 antibody (Catalogue No. 611738) and anti-RIPK1 clone 38 (Catalogue No. 610459) were from BD biosciences. Anti-caspase-8 1G12 was from Enzo (Catalogue No. ALX-804-447-C100). Anti-MLKL clone 3H1 was from EMD Millipore (Catalogue No. MABC604). Anti-IL-1β was from GeneTex (Catalogue No. GTX74034). Anti-RIPK3 antibody (Catalogue No. R4277), ATP and actinomycin D were obtained from Sigma. Ultrapure LPS, Pam3CSK4 and poly(I:C) were obtained from InvivoGen. zVAD was obtained from ApexBio. GSK'872 was obtained from Aobious. AP20187 was obtained from Clontech. CytoTox96 LDH-release kit was from Promega. All antibodies were used at 1/1,000–1/2,000 dilutions for western blot analyses.
9
Western Blot Analysis of IL-1β in THP-1 Cells
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THP-1 cells were harvested using radioimmunoprecipitation assay (RIPA) buffer and protein concentration of each sample was determined using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hertfordshire, UK). Equal amounts of protein were loaded on a 12% acrylamide gel and electrophoresed. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) according to the manufacturer’s instructions. The membrane was blocked for 1 h at room temperature with a 10% blocking solution (dry milk powder in TBST [tris-buffered saline + 0.1% Tween 20]). After blocking, the membrane was then incubated with anti-IL-1β (GeneTex, California, USA) or anti-β-actin (GeneTex) overnight at 4 °C before being washed with TBST and then incubated for 1 h at room temperature with the corresponding peroxidase-conjugated secondary antibody (Agilent Technologies, California, USA). Blots were washed again in TBST before being soaked in Pierce ECL Western Blotting Substrate (ThermoFisher) and exposed to x-ray film then the bands were developed.
10
Quantification of Cell Death Pathways
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To analyze cell death pathways induced in ESCRT-III-silenced cells, total (live and dead) supernatant and adherent cells were collected and counted. 1 × 106 cells were pelleted and lysed in 100 μL 2XSB buffer without DTT (4% SDS (#EU0660, Euromedex), 0.12 M Tris pH 6,8 (#10708976001, Sigma-Aldrich), glycerol 10% (#24388.295, VWR), dash of bromophenol blue (#B-5525, Sigma-Aldrich)) (1 million cells lysed per 100 μL buffer). When needed, DTT (#P2325, ThermoFisher) was added extemporaneously at 100 mM final concentration. Samples were immediately boiled for 5 min at 95°C, quickly spun and frozen at −20°C until use. The following antibodies were used for immunoblotting detection: anti-caspase-3 (#9662, Cell Signaling Technologies), anti-caspase-8 (#9429, Cell Signaling Technologies), anti-caspase-1 (produced in house – Peter Vandenabeele’s lab and Schotte et al. (2004) (link), anti-caspase-11 (#120–10454, Novus Biologicals), anti-cleaved Parp (#9544, Cell Signaling Technologies), anti-Gasdermin-D (Genentech), anti-IL-1β (#GTX74034, Genetex), anti MLKL (#MABC604, Millipore (reducing conditions) or #SAB1302339, Sigma-Aldrich (non-reducing conditions)), anti-phosphoMLKL (#ab196436, Abcam), anti-RIPK3 (#R4277, Sigma-Aldrich), anti-RIPK1 (#610459, BD Biosciences).
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