Western Blot Analysis of Tight Junction Proteins

Proteins were extracted from homogenized colonic tissues of mice using ice-cold RIPA lysis buffer containing complete EDTA-free protease inhibitor cocktail and PhosSTOP Phosphatase Inhibitor as previous (5 (link)). After centrifugation, the supernatants were collected, and protein concentrations were quantitated by BCA assay (Beyotime Biotech, Beijing, China). Forty-microgram protein of each sample were separated by 10% SDS-PAGE and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (BioRad, Hercules, CA, United States). Membranes were blocked with 5% defatted milk and incubated with indicated primary antibodies at 4°C for 12h. Then, membranes were incubated with HRP-conjugated anti-rabbit IgG secondary antibody (#7074, CST, Boston, MA, United States) at room temperature for 1h. Finally, the protein bands were test using Clarity™ ECL Western blot substrate (1705,060, Bio-Rad, Hercules, CA, United States) and captured using the ChemiDoc Touch imaging. Primary antibodies and dilutions used were anti–β-actin (AC026, ABclonal Biotechnology Co., Ltd., Wuhan, China, 1:50000), anti-ZO-1 (ab96587, Abcam, Cambridge, United Kingdom, 1:1000), and anti-occludin (ab167161, Abcam, Cambridge, United Kingdom, 1:1000).