Western blotting of tight junction proteins

Intestinal mucosal biopsies were frozen in liquid nitrogen until further use. Proteins were extracted from intestinal tissues, separated by SDS-PAGE, and then subsequently transferred onto PVDF membranes. The membranes were incubated with primary antibody rabbit anti-ZO-1(1:50, abcam), rabbit anti-occludin (1:250, abcam), or rabbit anti-β-Actin (1:5000, Beijing Zhongshan) over-night at 4°C, followed by exposure to secondary antibody (HRP- anti-rabbit IgG, 1:1000, GenScript) for 2 hours at room temperature. The proteins were visualized with an enhanced chemiluminescent detection system (Thermo) and exposed to X-ray film. Densitometry of the blots was performed using Quantity One 1-D analysis software (Bio-Rad).