Ripa lysis buffer for protein extraction

Snapped frozen liver tissues were treated with RIPA lysis buffer for protein extraction (Beyotime, Nanjing, China). Protein concentrations were determined using the BCA assay (Beyotime, Nanjing, China). Proteins (60 µg) were loaded into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature and incubated overnight at 4 °C with primary antibodies against NLRP3 (1:1,000; Cell Signaling Technology, USA), ASC (1:1,000; Santa Cruz, USA), Cleaved caspase1 (1:1,000; Santa Cruz, USA), Bcl-xl (1:1,000; Cell Signaling Technology, USA), Bax (1:1,000; Cell Signaling Technology, USA), Cleaved-caspase3 (1:1,000; Cell Signaling Technology, USA) and GAPDH (1:2,000; Proteintech, China). After 3 washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (1:10,000; Proteintech, China) at room temperature for 1 h. The proteins bands were developed using Electrochemiluminescence (Biorad, USA) reagents and imaged using the Imaging System (Peiqing, Shanghai, China). The gray value was quantified using Image J software.