Western blot analysis of p-p38MAPK and p38MAPK

Intestinal tissue was isolated and homogenized in lysis buffer (Beyotime Biotechnology, China) on ice. The homogenate was centrifuged (14 000 rpm, 15 min, 4°C) and the level of total supernatant protein was measured by BCA protein assay kit (Beyotime Biotechnology, China). The protein samples (20 μg) were boiled in Laemmle’s sampling buffer and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels. Next, the proteins were transferred to a polyvinylidene difluoride membrane and blocked with Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20. They were then incubated with anti-p38 MAPK or the anti-phospho-p38 MAPK antibody (1: 1000; Cell Signaling Technology, Danvers, MA) for 16 h at 4°C, followed by incubation with Alexa Fluor 680-conjugated anti-rabbit IgG (Molecular Probes). After washing 5 times for 5 min each time, the membranes were visualized by NBT/BCIP (Beyotime Institute of Biotechnology, Nantong, China). The protein bands were scanned and quantified using ImageJ software version 1.34 s (Wayne Rasband National Institute of Health) using β-actin for normalization.