Anti heme oxygenase 1 ho 1

Total proteins were extracted with lysis radioimmunoprecipitation assay buffer (Applygen, Beijing, China) and protease inhibitor cocktail (Applygen). The protein concentrations were determined by BCA assay (Beyotime, Shanghai, China). All steps were carried out on ice. Nuclear and cytosolic proteins were extracted using a commercial kit (KeyGEN BioTECH’s, Nanjing, China). The extracts were boiled in a metal bath at 95°C for 5 min. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out to separate the proteins. The proteins were then transferred to a polyvinylidene fluoride (PVDF; Solarbio) membrane for about 1.5 h. After blocking in 5% nonfat milk (Applygen) for 2 h, the PVDF membrane was incubated with the primary antibodies anti-Nrf2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-heme oxygenase-1 (HO-1; 1:500; Wanlei Biotechnology, Shenyang, China), anti-hypoxia-inducible factor-1α (HIF-1α; 1:500; BBI, Shanghai, China), anti-β-actin (1:5,000; abclonal, Wuhan, China), and anti-lamin B (1:500; abclonal) overnight at 4°C then followed by horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The membranes were washed three times for 10 min before obtaining protein bands by enhanced chemiluminescence reagents (Beyotime) and analyzed by ImageJ.

Proteins from the colon tissues of mice or Caco2 cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro transferred to PVDF membranes. Then membranes were blocked in 5% non-fat milk and incubated overnight at 4°C with the following primary antibodies: anti-signal transducer and activator of transcription 3 (STAT3) (WL01836; Wanleibio, Shenyang, China), anti-phosphorylated STAT3 (p-STAT3) (#9145; Cell Signaling Technology, Danvers, MA, United States), anti-IL-6 (WL02841; Wanleibio), anti-Keap-1 (WL03285; Wanleibio), anti-Nrf2 (WL02135; Wanleibio), anti-heme oxygenase 1 (HO-1) (WL02400; Wanleibio), anti-zonula occludens-1 (ZO-1) (WL03419; Wanleibio), anti-occludin (WL01996; Wanleibio), anti-claudin-1 (WL03073; Wanleibio), anti-OSM (PA5-81453; Invitrogen, Carlsbad, CA, United States), and anti-OSMR (ab210771; abcam, Cambridge, MA, United States); anti-β-actin (AT0001; Engibody Biotechnology, Inc., Milwaukee, United States) served as the internal control. Then membranes were incubated with the appropriate secondary antibody for 1 h at room temperature. The intensity of each band was scanned by the ChemiDoc™XRS+ Imaging System and analyzed with ImageJ software.