Moesin

Anesthetized mice were transcardially perfused with saline through the left ventricle. The brain was then removed and cerebral hippocampus was separated for extraction of protein. Protein concentrations were determined with a BCA Protein Assay Kit (Thermo Scientific). An equal amount of proteins was subjected to electrophoresis on SDS polyacrylamide gel, followed by electrotransfer to a nitrocellulose membrane. After blockage with 3% skimmed-milk powder in PBS with 0.1% tween-20 for 1 h, the membranes were probed overnight at 4°C with the primary antibodies specific for β-actin, claudin-5, occludin, ZO-1 (1:1000, Invitrogen, Camarillo, CA, USA), JAM-1 (1:200, Santa Cruz Biotechnology, Santa Cruz, USA), RAGE, RhoA, ROCK1, moesin, p-moesin (1:1,000, Abcam, Cambridge, UK), Src and p-Src (1:500, Cell Signaling, Beverly, Massachusetts, USA). After washing with TBST, the membranes were incubated with respective horseradish peroxidase-conjugated secondary antibodies (Beyotime, Shanghai, China) at a 1:3,000 dilution for 60 min at room temperature. Specific bands were visualized using enhanced chemiluminescence detection kit (Santa Cruz Biotechnology, California).

Monoclonal anti-ezrin antibody was purchased from LifeSpan Bioscience Inc. (Seatle, WA). Polyclonal anti-ezrin, ERM, phospho-ERM, β-actin and GAPDH antibodies were purchased from Cell Signaling (Danvers, MA). Goat anti-mouse podocalyxin antibody was purchased from R&D Systems (Minneapolis, MN). Polyclonal anti-Rho-GDIα antibodies were purchased from Sigma Aldrich (St. Louis, MO). Polyclonal anti-radixin, moesin, podocin and synaptopodin antibodies were purchased from Abcam (Cambridge, MA). Polyclonal anti-NHERF2 antibody was purchased from Thermo Fischer Scientific (Waltham, MA). Polyclonal anti-CLIC5 antibody was purchased from Alomone Labs (Jerusalem, Israel). For immunoblotting, all antibodies were diluted with Solution 1 (Can Get Signal; TOYOBO, Osaka, Japan) by a factor of 1:1000. For immunofluorescence analysis, all antibodies were diluted with Solution A (Can Get Signal; TOYOBO, Osaka, Japan) by a factor of 1:100.