Pab150009

Total protein was extracted using 1% Triton X-100 from the small intestine tissue. Total protein was quantified using bicinchoninic acid protein concentration assay kit (Bioswamp Life Science Lab) and proteins were separated by SDS-PAGE. The concentration of protein in each lane was 20 µg and 12% gel was used. The separated proteins were transferred onto polyvinylidene difluoride membranes and blocked with 5% non-fat milk in 2,4,6-trinitrobenzenesul-phonic acid (TNBS) for 2 h at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C: Anti-MLCK (1:5,000), anti-ROCK (1:5,000), anti-occludin (1:50,000), anti-ZO-1 (1:1,000), anti-p-MLC (1:1,000), anti-MLC (1:1,000) and anti-GAPDH (1:1,000). Following the primary antibody incubation, the membranes were washed three times for 10 min each in TNBS and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:1,000) at room temperature (goat anti-rabbit IgG, cat. no. PAB150011;goat anti-mouse IgG, cat. no. PAB150009, Bioswamp Life Science Lab) for 1 h. Protein bands were visualized using an electrochemiluminescence reagent (EMD Millipore) and a Tanon 5200 automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd.). Protein expression levels were analyzed using BandScan5.0 software (Glyko, Inc.).