Il 1β

The intestinal samples were lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitor cocktails (Sigma Co., St. Louis, MO, United States) for 10 min on a rocker at 4°C. Proteins of samples were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, Billerica, MA, United States). Each PVDF membrane was blocked with tris buffered saline tween (TBST; 100 mM Tris-HCl,150 mM NaCl, 0.05% Tween 20, pH 7.5) with 5% non-fat dried milk for 2 h, and then incubated with the following primary antibodies: NLRP3 (1:1,500), ASC (1:100), Caspase-1 (1:800), IL-1β (1:400), and β-actin (1: 5,000, Sungene biotech, China) overnight at 4°C on a shaker (SC390C, Shanghai Brave Construction Development co., Ltd., Shanghai, China). The goat anti-rabbit of 1:3000 (ZSGE-BIO, China) horse radish peroxidase (HRP) conjugate secondary antibody was then added and the WB results was observed through electrochemical luminescence (ECL) imaging system after adding the enhanced ECL reagent (Beyotime, Shanghai, China). The β-actin was used as a protein loading control. Quantitative analysis was carried out using Amersham Imager 600 (Cytiva, Montana, United States).

Proteins were extracted using RIPA buffer (Thermo, USA), according to the manufacturer's instructions, and the protein concentrations were measured using a BCA protein assay kit (Thermo, USA). An equal amount of protein from each sample was subjected to sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were blocked with 5% milk for 2 h at room temperature and incubated overnight at 4°C with primary antibodies, including TLR4 (1 : 1000), MyD88 (1 : 1000), NF-κBp65 (1 : 1000), phospho-IκBα (p-IκBα, 1 : 500), IL-1β (1 : 1000), TNFα (1 : 1000), and internal control β-actin (1 : 10000, Sungene Biotech Co., Ltd., Tianjin, China). Membranes were washed twice for 10 min in 1×TBST and then incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG: 1 : 12500; goat anti-mouse IgG: 1 : 12500, Sungene Biotech Co., Ltd., Tianjin, China) for 2 h. Membranes were then washed twice for 10 min in 1×TBST. Proteins were visualized by ECL (Millipore, USA), and blots were scanned in dark room. Densitometry analysis of bands was performed with the Image J software.