Western Blot Analysis of Protein Expression

Eight percent to 12% SDS-PAGE gels was electrophoresed with some amounts of protein per lane (20 μg) based on our established protocol [16 (link), 29 (link)]. Proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk in TBST for 1 h at room temperature. Thereafter, the PVDF membranes were incubated with different primary antibodies, including rabbit anti-Occludin-1 (1:1000, Invitrogen), rabbit anti-MMP-9 (1:1000, Abcam), rabbit anti-β-catenin (1:2000, Abcam), rabbit anti-p-β-catenin (1:1000, Abcam), rabbit anti-GSK-3β (1:2000, Abcam), rabbit anti-Caspase-3 (1:2000, ABclonal), rabbit anti-GAPDH (1:3000, Abcam), mouse anti-Claudin-5 (1:1000, Invitrogen), and rat anti-ZO-1 (1:500, Santa Cruz Biotechnology). After washing with TBST, the PVDF membranes were treated with HRP-conjugated secondary antibody (1:5000) for 1 h at room temperature. Immunolabeling was probed with enhanced ECL kit (Biosharp). The chemiluminescence level can be captured with the imaging system, and the data were normalized to GAPDH.