Protein Expression and Phosphorylation Analysis

To test the protein level of tight junction protein (ZO-1, occludin and claudin-1), the phosphorylation level of the proteins, which belongs to the downstream of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathway, were tested. The tissue samples were homogenized by using Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease inhibitor and phosphatase inhibitor cocktail (Songon Biotech, Shanghai, China). The concentration of protein was determined by using the BCA protein assay kit. The tissue sample was mixed with 5 × loading buffer (Sangon Biotech, Shanghai China) and heating at 100^∘C for 5 min. The SDS-PAGE gel kits were purchased from Sangon Biotech (Shanghai, China). The sample (50 μg protein) was loaded to each well. The detailed process was described in the previous study (Zhai et al., 2018 (link)). The primary antibodies including β-actin, p38, p-p38, c-Jun, p-c-Jun, p65, p-p65, and ZO-1, claudin-1 were purchased from Cell signaling Technology (CST, Danvers, MA, United States) and occludin was purchased from Thermo Fisher Scientific (Waltham, MA, United States).