Df6252

The inferior lobe of the lung tissues was homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Inc.). After that, 30 μg protein was separated in 10% SDS-PAGE. Proteins were then transferred to PVDF membranes (Millipore). Then, the PVDF membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies against p-P65 (1 : 1000, AF2006, Affinity); P65 (1 : 1000, AF5006, Affinity); p-IKBα (1 : 1000, AF2002, Affinity); IKBα (1 : 1000, AF5002, Affinity); NLRP3 (1 : 1000, DF7438, Affinity), ASC (1 : 2000, sc-514414, Santa Cruz Biotechnology, INC), Caspase-1 p20 (1 : 2000, AF4005, Affinity), Pro-GSDMD (1 : 1000, AF4012, Affinity), GSDMD p30 (1 : 1000, DF12275, Affinity), IL-18 (1 : 1000, DF6252, Affinity), IL-1β (1 : 1000, AF5103, Affinity), and β-actin (1 : 5000, AF7018, Affinity) at 4°C overnight. Then, the membranes were then incubated with the appropriate secondary antibodies at room temperature for 2 h. β-actin was selected as the loading control. Finally, the protein bands were caught by using a Chemiluminescence image analysis system (Tanon, Shanghai, China) and analyzed with the Image J software (National Institutes of Health, USA).

Berberine (BBR, purity ≥ 98%) was supplied by Meilun Biotechnology Co., Ltd. (Dalian, China). Uric acid (UA, purity ≥ 99%) was acquired from Sigma-Aldrich (St. Louis, MO, USA). Z-YVAD-FMK (ZY) was obtained from ApexBio (Houston, USA). Enzyme-linked immunosorbent assay (ELISA) kits for human IL-1β and IL-18 were supplied by Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). The LDH assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against Caspase1 (DF6148), IL-1β (AF5103), IL-18 (DF6252), and β-actin (AF7018) were purchased from Affinity Biosciences (Cincinnati, OH, USA). Antibodies against NLRP3 (ab263899), GSDMD (ab210070) were acquired from Abcam (Cambridge, UK). Antibodies against ASC (WL02462), cleaved-Caspase1 (cl-Caspase1, WL03450), cl-Caspase3 (WL02117), cl-Caspase9 (WL02117), BAX (WL01637), and BCL-2 (WL01556) were supplied by Wanshi Biological Technology Co., Ltd. (Shenyang, China). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from EarthOx, LLC (CA, USA). And siRNA was offered by General Biological Systems Co., Ltd. (Anhui, China). Lipofectamine®²⁰⁰⁰ Reagent was obtained from Invitrogen Co., Ltd. (Carlsbad, CA, USA). Opti-MEM^® I Reduced Serum Medium was offered by Gibco Co., Ltd.