Anti p p65

Samples were lysed in RIPA buffer lysis containing protease inhibitors (PMSF) (Beyotime, China) at 4 °C, and used bicinchoninic acid (BCA) assay to determine the concentration. To further dilute the sample, 6× SDS-PAGE loading buffer was added and boiled (100 °C) for 10 min. An equal amount of the samples (20 μg) was acceded, electrophoresed on a 10% SDS-polyacrylamide denaturing gel, and then transferred to a polyvinylidene fluoride (PVDF) membrane. Anti-HMGB1 (1:1000; Servicebio, Wuhan, China), anti-p65 (1:1000; Bioss, Beijing, China), anti-p-p65 (1:1000; Sigma, Ronkonkoma, NY, USA), anti-TLR4 (1:1000; Sigma, USA), anti-MYD88, anti-NLRP3, anti-IL-1β, anti-pro-caspase-1 (1:500; Wanleibio, Shenyang, China), anti-ASC (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (1:5000; Bioss, China) as the primary antibody. With GAPDH as a reference, and the protein was measured and analyzed by Image J 1.48V and Image Lab 4.0 Software (Bio-Rad, Hercules, CA, USA).

Lysing of the cells was conducted in a cell lysis buffer (Beyotime, Shanghai, China) that contained 0.2 μM leupeptin, 1.5 μM pepstatin A, and 1 μM phenylmethylsulfonyl fluoride. The 5% nonfat milk was utilized to block proteins loaded onto a nitrocellulose membrane after they had been separated on an SDS-denatured polyacrylamide gel, followed by the incubation of membranes at 4°C over the night with rabbit primary antibodies (anti-HS3ST1, anti-cleaved caspase-3, anti-caspase-3, anti-cleaved caspase-8, anti-caspase-8,anti-GAPDH, anti-Bax, anti-Bcl-xl, anti-SPOP, anti-FADD, anti-p-p65, and anti-p65, Sigma-Aldrich, St. Louis, MO, USA). The following day, the membranes were rinsed before being incubated with secondary antibody (Sigma-Aldrich, St. Louis, MO, USA). Visualization was achieved utilizing a chemiluminescence enhanced chemiluminescence (ECL) western blotting analysis system (B&D, San Jose, CA, USA). After being normalized to GAPDH, the protein levels were determined utilizing ImageJ software (NIH, USA).