2.9 Western blotting
The protein was extracted from colonic tissues and cells with RIPA reagent containing protease inhibitors and phosphatase inhibitors (CWbio, Jiangsu, China), and the protein concentration was detected by BCA protein determination kit. The same amount of protein was separated on 8% SDS-PAGE gel and then transferred to PVDF membrane. Blocked with 5% bovine serum albumin (Solarbio, Beijing, China) at room temperature for 2 hours and incubated with different antibodies at 4 oC overnight. The membrane was washed with TBST for 3 times and incubated with secondary antibody (Affinity, Jiangsu, China) at room temperature for 1 hour. The film was observed by chemiluminescence kit. The protein was visualized using an ECL kit (Biosharp, Anhui, China) and quantitatively analyzed using Image J software. The sequences of primers are listed in Table S1.