Anti nlrp3

After different treatments, the BV-2 cells were washed with cold PBS twice, and the cells were fully lysed with RIPA lysate containing 1% protease inhibitor. The lysate was collected and centrifuged at 4 °C at 12,000 rpm for 25 min. Part of the supernatant was taken for protein-concentration determination, and the remaining supernatant was mixed with the loading buffer and boiled at 100 °C for 10 min. Fifty μg of protein were injected into the pore of 12% and 8% SDS polyacrylamide gel to isolate proteins with different molecular weights. The protein was then transferred at a constant voltage to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was immersed in TBST solution containing 5% skim milk and slowly shaken at room temperature for 1 h. Wash the protein band with TBST three times. Anti-β-actin (1:1000) (Affinity, Changzhou, China), Anti-COX2 (1:1000) (Bioss, Beijing, China), anti-NLRP3 (1:1000) (GeneTex, Southern California, CA, USA), and anti-Caspase-1 (1:500) (Wanleibio, Shenyang, China) antibodies were used to incubate the protein bands at 4 °C overnight. On the second day, protein bands were incubated with either goat anti-rabbit (1:10,000) or goat anti-mouse secondary antibody (1:10,000) (ZSGB BIO, Beijing, China) for 1 h. All protein bands were measured by ECL Western blot assay.