Anti il 1β antibody

Treated cells were lysed in RIPA containing protease inhibitor (PMSF, 1 : 100) on ice for 30 minutes. The cell lysate was then centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was then transferred to new centrifuge tubes. The protein concentrations were determined using a BCA kit (Beyotime Biotech, Shanghai, China); equal amounts (40 μg) of protein samples were separated using SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk powder in TBST for 2 h. The membranes were probed overnight at 4°C with anti-NLPR3 (1 : 1000, Abcam, UK), anti-ASC (1 : 1000, Proteintech, IL, USA), anti-Caspase1 (1 : 1000, Proteintech, IL, USA), and anti-IL-1β antibodies (1 : 1000, Proteintech, IL, USA). Further, the membranes were incubated with HRP-conjugated secondary antibodies(1 : 2000, Proteintech, IL, USA) for 2 h at room temperature. The antibody binding was detected using the enhanced chemiluminescence (ECL) detection kit (Beyotime Biotech, Shanghai, China).

Tissue samples were prepared and preserved through paraffin embedding and then dewaxed and blocked in a hydrogen peroxide/methanol solution. Antigen retrieval was performed using sodium citrate (pH 6.0) for 2×20 min at 80°C. After that, the sections were incubated with anti-SREBP2 antibody (Abcam, Cambridge, UK) and anti-IL-1β antibody (Proteintech, Chicago, USA) overnight at 4°C, followed by incubation with HRP-conjugated anti-rabbit IgG secondary antibody (Abcam) at 37°C for 30 min. The sections were stained with DAB and counterstained with hematoxylin, dehydrated in ethanol, mounted in dimethylbenzene, and placed under a coverslip. Analysis of IHC images was performed using ImageJ (NIH, Bethesda, USA).