Anti il 1β

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).

Equal amounts of protein samples (28 μg) were subjected to a SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk in TBST for 2 h followed by incubation with the following primary antibodies as follows: anti-p-NF-κB P65 (1:1000, Cell Signaling Technology), anti-NF-κB P65 (1:1000, Cell Signaling Technology), anti-Iba-1 (1:1000, Abcam), anti-β-actin (1:7500, Bioss antibodies), anti-GR (1:1000, Bioss antibodies), anti-NLRP3 (1:1500), anti-ASC (1:500), anti-pro-caspase 1 (1:500), anti-caspase-1 p20 (1:750), anti-pro-IL-1β (1:10000), anti-IL-1β (1:750), anti-IL-18 (1:1000) and anti-Lamin B (1:750) both from Wanlei Biotechnology (Shenyang, China). After three times washing with TBST, the membranes were incubated with appropriate secondary antibody. The protein bands were visualized by the ECL detection system (Thermo Scientific, Waltham, MA, United States) and quantified using Image J software. Nuclear and Cytoplasmic Protein Extraction Kit (Applygen Technologies, Co., Ltd., Beijing, China) was used to extract the cytoplasmic/nuclear proteins of NF-κB P65 according to the manufacturer’s protocol.