Anti caspase 1

LPS (Sigma #L4391), adenosine 5'-triphosphate disodium salt hydrate (ATP) (Sigma #A6419), N-acetyl-L-cysteine (NAC) (Glpbio #GC11786) and vitamin C (Glpbio #GC12979) were dissolved in sterile deionized water. The AKT inhibitor MK-2206 (Glpbio #GC16304) was dissolved in dimethyl sulfoxide (DMSO). Anti-NLRP3 (#15101), anti-GSDMD (#93709), anti-phospho-AKT (#4060), anti-phospho-mTOR (#5536) and anti-β-actin (#3700) were obtained from Cell Signalling Technology. Anti-caspase-1 (#PA5-78915) was purchased from Invitrogen. Anti-Nox4 (ab133303) was obtained from Abcam.

After different treatments, the cells were incubated with RIPA lysis buffer containing a protease inhibitor cocktail on ice for 10 min. Cell lysates were then harvested by scraping, followed by brief sonication and centrifugation at 12,000 × g for 10 min at 4 °C. The protein concentration was determined via the BCA assay. Equal amounts of protein were separated by SDS/PAGE and the bands were electroblotted onto PVDF membranes. The membranes were blocked with 5% non-fat milk dissolved in TBST and then incubated with appropriate primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-NLRP3 (1:1000, Abcam), anti-Gasdermin D-N-terminal (1:1000, Invitrogen), anti-caspase-1 (1:1000, Invitrogen), anti-cleaved caspase-1 (1:1000, Invitrogen), anti-GAPDH (1:10000, Abcam), α-Fodrin (1:1000, Cell Signaling Technology), anti-ATP5A1 (1:1000, Invitrogen), Calpastatin (1:500, ABclonal), and Flag (1:1000, Abcam). The membranes were then incubated with the following horseradish peroxidase-conjugated secondary antibodies: HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG purchased from Proteintech (1:5000, America). The immunofluorescent bands were then visualized and quantified using an ECL chemiluminescence kit (Absin, Shanghai, China) and a chemiluminescence–western blotting detection system (Tanon, Shanghai, China).