Cells were seeded in six-well plates at a density of 5 × 105 cells/well. Cells were washed twice with cold PBS, then detached from the plate surface with a cell scraper. Following mixture with 200 μL RIPA (containing 1% protease inhibitor), cells were incubated on ice for 20 min. Supernatants were collected after high-speed centrifugation (12000 rpm, 15 min); they were then snap-frozen in liquid nitrogen and stored at − 80 °C. Protein concentrations were determined by a Bicinchoninic Acid Protein Assay Kit (CWBIO, Beijing, China). Western blots were conducted as previously described11. In brief, equal amounts of protein from each sample were resolved in loading buffer and boiled for 5 min. The proteins (30 μg/lane) were separated on a sodium dodecyl sulfate polyacrylamide gel, then electrotransferred to polyvinylidene difluoride membranes. The blots were washed twice with Tris-buffered saline plus Tween (TBST; 20 mM Tris [pH 7.4], 0.9% NaCl, and 0.1% Tween-20), blocked with 5% bovine serum albumin in TBST for 2 h, and incubated with primary antibodies: rabbit anti-NLRP3 (1 μg/ml) and rabbit anti-caspase-1 (3 μg/ml) (Novus Biologicals, USA) at 4 °C overnight. Following three washes with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000, Abcam) at room temperature for 1.5 h. Signals were detected with chemiluminescence reagents (Millipore, USA) and imaged by a Multispectral Imaging System (Biospectrum AC Chemi HR 410, UVP, CA, USA). An anti-GAPDH antibody (1:5000, CST, MA, USA) was used as an internal standard. Optical densities of target proteins were quantified by ImageJ and normalised to the density of GAPDH.

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