Anti caspase 1

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.

Immunostaining of knee joint sections was performed with specific antibodies against target proteins according to a previously published protocol with some modifications (Wang et al. 2019 (link)). Sections were incubated with anti-NLRP3 (1:100), anti-ASC (1:100), anti-caspase-1 (Wanleibio, 1:100), anti-GSDMD (Affinity Biosciences, 1:100), and anti-IL-1β (1:100) antibodies overnight at 4 °C. On the following day, these tissue sections were incubated with a polymer-HRP detection system (PV9001, ZSGB-BIO) and visualized with a diaminobenzidine (DAB) peroxidase substrate kit (ZLI-9017, ZSGB-BIO). Each section was evaluated under a microscope (DMi8, LEICA) at 20 × magnification. Histological analyses were performed in a blinded manner by two independent observers. Image-ProPlus6 software 6.0 (Media Cybernetics, Inc.) was used to analyze the average integrated optical density (IOD) of three randomly selected areas of the acquired images.