Cav-1, claudin-5, and iNOS in total cell lysates and subcellular fractions (CF, MF, and ACF) were analyzed by Western blot as we described previously [16 (link)]. In brief, protein samples were electrophoresed in SDS-PAGE acrylamide gels, transferred onto nitrocellulose membranes (Bio-Rad). After blocking with 5 % nonfat milk, membranes were incubated overnight at 4 °C with primary antibodies against Cav-1 (Santa Cruz Biotech, 1:500), claudin-5 (Invitrogen, 1:1000), or iNOS (Invitrogen, 1:500), followed by incubation with corresponding HRP-conjugated anti-rabbit or anti-mouse antibodies (Santa Cruz Biotech, 1:1000). The membranes were developed with the SuperSignal West Pico HRP substrate kit (Pierce) and photographed on a Kodak 4000 image station (Carestream Molecular Imaging). To control sample loading and protein transfer, the membranes were stripped and reprobed with β-actin antibody (Santa Cruz Biotech, 1:1000). For subcellular fraction samples, we noticed that the actin levels were comparable for each sample among different fractions, so we only used one normalizing loading control (CF actin) for all three subcellular fractions.