Nf κb

Jejunum tissues were homogenized at 4°C in RIPA lysis buffer which contained protease inhibitors (PMSF) (Beyotime, Shanghai, China), and concentrations were determined using a BCA assay (35 (link)). Samples were further diluted, and 5× SDS-PAGE loading buffer was added and boiled for 5 min. Equal amounts of protein (10 μg) were loaded onto 12% SDS-polyacrylamide denaturing gels before being transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with tris-buffered saline Tween (TBST) containing 5% non-fat milk powder for 1 h at room temperature, the membrane was incubated overnight with diluted primary antibodies against ZO-1 (1:1,000; ABclonal, Wuhan, China), occludin (1:1,000; Selleck Chemicals, USA), claudin-1 (1:1,000; ABclonal, China), TLR4 (1:500; Proteintech, Wuhan, China), MyD88 (1:500; Wanleibio, Shenyang, China), NF-κB (1:500; Wanleibio, China), NLRP3 (1:1,000; Wanleibio, China), ASC (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), caspase-1 (1:500; Wanleibio, China), IL-1β (1:500; Wanleibio, China), IL-18 (1:1,000; Wanleibio, China), and GAPDH (1:5,000; Proteintech, China). Electrochemiluminescence liquid (ECL) (Tanon, Shanghai, China) was used to detect the signal. ImageJ software was used to assess protein levels. The target protein levels were normalized to GAPDH, and the radioactivity was compared with the control group.

Oridonin (>98%) was purchased from National Institutes for Food and Drug Control of China. Adriamycin was purchased from Pharmacia Italia S.p.A., Gaggiano, Italy. Dimethyl sulfoxide (DMSO), trypsin, Tris, glycine, acrylamide, methylene diacrylamide were purchased from Sigma‐Aldrich. RPMI‐1640 cell culture medium was purchased from Gibco. Fetal bovine serum (FBS) was obtained from Sijiqing Hangzhou Bio Engineering Co., Ltd. Antibodies for caspase‐8, caspase‐3, caspase‐7, MEKK1, MEK7, JNK, IKBα, NF‐κB, AR, TGF‐β, Smad 2/3, TNF‐α, STEAP, Smad‐4, β‐actin and the secondary antibodies were purchased from Wanleibio.