Anti cleaved caspase 1 89332

Total cellular proteins were prepared using ice-cold RIPA buffer containing 1% halt protease inhibitor (Beyotime). Proteins (50 µg/sample) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk followed by incubation with primary anti-IL-6 (ab214429), anti-TNF-α (ab183218), anti-IL-1β (ab216995), anti-IL-18 (ab207324), anti-NLRP3 (ab270449), anti-ASC (ab155970), anti-GSDMD-N (ab215203), anti-β-actin (ab8226) (1:1,000, Abcam, USA), and anti-cleaved caspase-1 (#89332) (1:1,000, Cell Signaling Technology, USA) antibodies at 4 ℃ overnight. The membranes were washed and incubated with anti-mouse (ab97040) and anti-rabbit (ab7090) HRP secondary antibodies (1:2,000, Abcam), and the protein bands were detected with enhanced chemiluminescence (Bio-Rad).

MCC950 (M6164) was purchased by AbMole BioScience (Houston, USA). DSS (D122354, MW 40000) was purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Anti-β-actin (A3854) was purchased from Sigma (St. Louis, USA). Anti-NLRP3 (ab214185), Anti-IL-1β (ab9722), anti-interleukin-6 (IL-6, ab9324), and Anti-Iba1 (ab5076) were purchased from Abcam (Cambridge, USA). Anti-cleaved caspase-1 (89332) was purchased from Cell Signaling Technology (Beverly, USA). Anti-ASC (SC-514414) was purchased from Santa Cruz Biotechnology (Dallas, USA). Anti-tumor necrosis factor-alpha (TNF-α, AF8208) was purchased from Beyotime Biotechnology (Shanghai, China). Malondialdehyde (MDA, A003-1-2), catalase (CAT, A007-1-1), and superoxide dismutase (SOD, A001-3-2) assay kits were purchased from Nanjing Jiancheng Bioengineering (Nanjing, China).