The culture supernatants were collected and concentrated as previously described (Zhao et al., 2021 (link)). Cell pellets were washed in ice-cold PBS and lysed with RIPA containing the protease inhibitor of phenylmethanesulfonyl fluoride (PMSF, 1 mM). The protein samples were quantified with a BCA Protein Assay Kit (Thermo Scientific, USA) according to the manufacturer’s protocol. Each sample (30 μg) of cell lysates or cell supernatants (10 μl) were separated by 12% SDS-PAGE, and the electrophoresis protocol was set as follows: 80 V 1 h 05 min and 120 V 30 min. Then, the protein was transferred onto 0.22 or 0.45 μm PVDF membrane (Millipore, USA) under conditions of 200 mA/1 h or 1 h 30 min at 4°C. The membranes were blocked in 5% bovine serum albumin (BSA) in PBST for 2 h at room temperature and incubated at 4°C overnight with different primary antibodies (Abs), including IL-1β (1:1,000), caspase-1 (1:1,000), NLRP3 (1:1,000), and β-actin (1:5,000). The HRP-conjugated secondary antibodies were rabbit anti-goat IgG (H+L) (1:5,000) or goat anti-mouse IgG (H+L) (1:5,000, EarthOx, USA). The blots were developed using the ChemiScope Western Blot Imaging System (Clinx Science Instruments, China).
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