Western Blot Analysis of Endothelial and Pericyte Proteins

The expression levels of TJ proteins and transporter proteins were analyzed by Western blotting [20 ]. After the examination of TEER, Western blots were performed to analyze protein extracts from both endothelial cells and pericytes. These extracts were obtained by lysing the cells with sample buffer (62.5 mM Tris, 2% SDS, 10% glycerin, 0.0125% bromophenol blue, pH 6.8) and homogenizing them on ice. The lysates were resolved by SDS‒PAGE and transferred to PVDF membranes. The membranes were incubated overnight in BlockAce blocking solution at 4 °C. Then, the membranes were incubated with primary antibodies for 1 h at 25 °C. After being washed three times, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG antibody (1:5000) for 1 h at 25 °C. The membranes were then washed three times, and signals were visualized with a LAS3000 chemiluminescence detector (Fujifilm Co., Tokyo, Japan). We confirmed that the bands matched the molecular weights of the specific proteins of interest, i.e., CD31 (120 kDa), ZO-1 (225 kDa), Claudin-5Claudin-5 (24 kDa), P-gp (180 kDa), BCRP (65–80 kDa), Glut1 (40–60 kDa), TfR (90 kDa), and b-actin (42 kDa).