Anti il 1β nb600 633

Proteins were extracted from cells or tissues using RIPA buffer (Thermo Fisher Scientific, 89900) with protease inhibitor cocktail (Sigma-Aldrich, S8820). Protein quantification was done using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Thirty microgram total protein was loaded onto 4%–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, 4561094) for electrophoresis. The following antibodies were used for protein detection: anti-α-Tubulin antibody (#2144, 1:1000) was purchased from Cell Signaling Technology. Anti-ASC antibody (sc-514414, 0.4 μg/ml) and anti-caspase-1 antibody (sc-1218, for pro-caspase-1 and p20, 0.5 μg/ml) antibody were purchased from Santa Cruz Biotechnology. Anti-NLRP3 (ab214185, 1:500), anti-NLRC4 (ab99860, 1:1000) and anti-AIM2 (ab180665, 1:1000) antibodies were purchased from Abcam. Anti-IL-1β (NB600-633, for pro-IL-1β and mature IL-1β, 1:1000) antibody was purchased from Novus Biologicals. Optical density was analyzed on a The ChemiDoc XRS+ system (Bio-Rad). The specificity of antibody staining was shown in Supplementary Figure S2.

The following antibodies were used for immunohistochemistry: rabbit polyclonal anti-TNF-α (#ab6671) and rabbit polyclonal anti-vascular endothelial growth factor (VEGF) (EP1176Y, #ab52917) antibodies were from abcam; rabbit polyclonal anti-IL-1β (#NB600-633) antibody was from Novus Biologicals. The following antibodies were used for flow cytometry: rat monoclonal APC anti-CD45R (B220) (RA3-6B2, #17-0452-83), mouse monoclonal PE anti-CD95 (APO-1/Fas) (15A7, #12-0951-81), mouse monoclonal PerCP-Cyanine5.5 anti-CD19 (SJ25C1, #45-0198-42), rat monoclonal FITC anti-CD5 (53-7.3, #11-0051-82), rat monoclonal PE anti-CD1d (1B1, #12-0011-82), rat monoclonal APC anti-IL-10 (JES5-16E3, #17-7101-82) antibodies were from Thermo Fisher Scientific; rat monoclonal PE-Cy™7 anti-CD19 (ID3, #552854) antibody was from BD Biosciences; Alexa Fluor® 488 anti-GL7 (GL7, # 144612) antibodies was from BioLegend. The following antibodies were used for confocal microscopy: rat monoclonal Alexa Fluor® 488 anti-CD4 (GK1.5, #100423) and rat monoclonal APC anti-CD25 (PC6, #102012) antibodies were from BioLegend; rat monoclonal PE anti-IL-17A (eBio17B7, #12-7177-81) antibody was from Thermo Fisher Scientific; mouse monoclonal PE anti-STAT3 (pY705) (4/P-STAT3, #612569) antibody was from BD Bioscience; rabbit polyclonal PE anti-Foxp3 antibody (#NP100-39002) antibody was from Novus Biologicals.

Antibodies: anti-IL-1β (NB600-633, Novus Biologicals, Littleton, CO, USA); anti-HA (Ab137838, Abcam, Cambridge, UK); anti-Flag (F1804, Sigma-Aldrich, St Louis, MO, USA); anti-NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA); anti-ASC (AG-25B-0006-C100, Adipogen, San Diego, CA, USA)
Reagents: recombinant human IL-1β (IL038, Sigma-Aldrich, St Louis, MO, USA), recombinant human TNF-α (ab9642, Abcam, Cambridge, UK); Z-YVAD-FMK (S8507, Selleck, Shanghai, China); Z-VAD-FMK (S7023, Selleck, Shanghai, China); MCC950 (S8930, Selleck, Shanghai, China); Bay 11-7082 (S2913, Selleck, Shanghai, China); IL-1R antagonist (sc-221747, Santa Cruz Biotechnology, Santa Cruz, CA, USA).