Anti occludin 1

In brief, intestinal mucosal samples were homogenized in 1 mL lysis buffer and centrifuged at 12,000×g at 4°C for 30 min for supernatants collection, and then the protein concentrations were measured using phenol reagent method. An equal amount of proteins samples (20 to 40 μg) was separated on a 10% or 12% reducing polyacrylamide gel and transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies. Immunoblots were blocked with 3% bull serum albumin (BSA) in Tris-buffered saline for 70 min at room temperature and incubated overnight at 4°C with the specific primary antibodies in Tris-buffered saline and 0.05% Tween 20 containing 1% BSA. The specific primary antibody included rabbit anti-claudin-1 (1:1,000; Proteintech, Chicago, IL, USA) and anti-occludin-1 (1:1,000; Proteintech, USA). Blots were washed with the same buffer and then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; Proteintech, USA) for 120 min at room temperature. Membranes were washed in the same buffer and antigen-antibody complexes visualized by using an ECL kit (Pierce, Waltham, MA, USA). The density of the bands was quantified using the Image J 1.46r analysis software.