Chemidoc touch imaging

The proteins were resolved by gel electrophoresis and transferred onto the nitrocellulose membrane. The blots were blocked using blocking solution (5% skim milk in 1× TBS). The primary antibodies were made in 1% skim milk (prepared in 1× TBS) and added onto the blots and were left for overnight incubation in shaking conditions at 4 °C. Next day, two washes were given using 0.1% TBST (0.1% Tween-20 in 1× TBS) for 5 min each. The secondary antibodies prepared in 1% skim milk was added to the blots and left for shaking for 1-h incubation at room temperature. 2–3 washes were given with 0.1% TBST before developing the blot using Millipore Immobilon Forte Western HRP Substrate kit (Cat number-WBLUF0100) in the Biorad Chemidoc Touch imaging system.
For stripping of proteins from the blot, 1X Millipore Reblot Plus Strong Antibody Stripping Solution (Cat number-2504) was added to the blot and left for shaking at room temperature for 20–25 min. The blot was developed using the ECL kit to check for any signal on the membrane. Two washes were then given with blocking solution (5% skim milk in 1× TBS) for 5 min each and then proceeded with incubation with the primary antibody solution.

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).