After treatments, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in radio immunoprecipitation assay buffer (Shenneng Bocai Biotechnology Co., Ltd., Shanghai, China) containing 1 mM phenylmethanesulfonyl fluoride and 1× phosphatase inhibitor cocktail (Roche, Mannheim, Germany). Protein was extracted, and equal amounts of protein (30 μg per lane) were separated by 8% or 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore). Blots were probed with primary antibodies overnight at 4°C. After membranes were washed, horseradish peroxidase-conjugated secondary antibody (Proteintech Group, Chicago, IL, USA) was applied, and proteins were visualized by using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). The following primary antibodies were used: anti-occludin and anti-ZO-1 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), anti-claudin-1 and anti-β-actin antibodies (Abcam, Cambridge, MA, USA), anti-phospho-p38 MAPK (Thr180/Tyr182), and anti-p38 MAPK antibodies (Cell Signaling Technology). Band intensity was analyzed with Quantity One version 4.6.2 software (Bio-Rad) and compared with the internal standard β-actin.