Pro il 1β

After the proteins were separated by electrophoreses, all samples were shifted to polyvinylidene difluoride membranes (Millipore Corporation, Massachusetts, USA). The membranes were incubated with the first Abs, NF-κB p65 (1:500), p-NF-κB p65 (1:500), TNF-α (1:500), Pro-IL-1β (1:1000), IL-6 (1:1000), IL-10 (1:500), Bad (1:750), Bcl-2 (1:500), Cyt C (1:1000), Cleaved-caspase 9 (1:500), Cleaved-caspase 3 (1:500), Cleaved-caspase 8 (1:1500), PI3K p110 (1:1000), AKT (1:500), p-AKT (1:500), MAP4 (1:500) (Wanlei Biotechnology Corporation, Shenyang, China), for 12~16 h at 4°C and then with the HRP conjugated anti-rabbit Ab (1:4000) (Wanlei Biotechnology Corporation, Shenyang, China) for 90 min at room temperature. Western blotting was monitored by using a chemiluminescence detection reagent (Meilun Biotechnology Corporation, Dalian, China) and visualized using Tanon 5200 Automatic Gel Imager (Tanon Science & Technology Corporation, Shanghai, China). For quantification, β-Tubulin (1:500) was used as the inner standards of tissue proteins.

Alanine amiotransferase (ALT), triglyceride (TG) and total cholesterol (TC) assay kits were purchased from Nanjing Jiancheng Biotechnology (Nanjing, Jiangsu, China). RNAios Plus, Pri-meScript™ RT reagent kit, and TB Green® Pre-mix Ex Taq™ II were obtained from Takara Biotechnology (Tokyo, Japan). The Caspase-1 Activity Assay Kit and Hoechst 33342/Propidium Iodide (PI) Double Staining Kit were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade and commercially available.
The following primary antibodies were used: mouse anti-GAPDH (6C5) antibody from Beyotime (Shanghai, China), mouse anti-ASC/TMS1/PYCARD (F-9) antibody from Santa Cruz (Santa Cruz, CA), mouse anti-beta-actin (2D4H5) antibody from Proteintech (Wuhan, China), rabbit polyclonal antibody against GSDMD from Proteintech (Wuhan, China), and rabbit anti-cleaved N-terminal GSDMD (EPR20829-408) antibody from Abcam (Cambridge, UK). The rabbit polyclonal antibodies against NLRP3, pro caspase-1, caspase-1 p20, pro IL-1β, IL-1β, and IL-18 were obtained from Wanleibio (Shenyang, Liaoning, China).