Jam 1

The total proteins of brains were extracted by icecold cell lysis buffer (Beyotime, Shanghai, China). Bicinchoninic acid assay protein assay kit (Beyotime) was used to determine the total protein content. Equal amount of protein preparations were run on 8%-12% sodium dodecyl acrylamide-polyacrylamide gels, electro-transferred topolyvinylidine difluoride membranes, blotted overnight with primary antibodies and reacted with appropriated peroxidase-labelled secondary antibodies (Zhongshan Biotechnology Co., Beijing, China). Immunoreactive proteins were detected by the chemiluminescence assay (ECL Millipore, Billerica, Massachusetts, USA) using LAS-4000 chemiluminescence imaging system (Fujifilm, Tokyo, Japan). Quantitative analysis was performed using AlphaEasaFC analysis software (Alpha Innotech, San Leandro, California, USA) with horseradish peroxidase-conjugated monoclonal antibody against -actin serving as a control. 23 The primary antibodies included occludin (1:1000; Epitomics, Burlingame, California, USA), JAM-1 (1:1000; Cell Signalling, Danvers, Massachusetts, USA), AQP4 (1:1000; Cell Signalling), MMP-9 (1:1000; Santa Cruz Biotechnology, Santa Cruz, California, USA) and -actin (1:5000; Anbo Biotechnology Co., Ltd, San Francisco, California, USA).