Anti claudin 3

At 72 hours after GMH, following transcardial PBS perfusion, ipsilateral hemispheres were collected and processed as previously described (Huang et al., 2012 (link)). Homogenates were separated by SDS-PAGE and transferred onto nitrocellulose membranes, which were incubated with the following primary antibodies: anti-phospho-Akt (Ser473), anti-Akt (both from Cell Signaling Technology, Beverly, MA, 1:2000), anti-ZO1, anti-IL-17 (both from AbCam, Cambridge, MA, 1:1000), as well as anti-occludin and anti-claudin-3 (both from Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000). GTP-Rac1 and total-Rac1 were detected using Rac1 Activation Assay Kits (Millipore, Temecula, CA). Secondary antibodies consisted of goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (both from Santa Cruz Biotechnology, 1:5000, sc-2005 and sc-2004 respectively). Immunoblots were visualized with the Amersham ECL Prime Western Blotting Detection Reagent (GE Life Sciences, Piscataway, NJ) and semiquantitatively analyzed using Image J software (4.0, Media Cybernetics, Silver Springs, MD). Results are expressed as relative density ratio, normalized to mean density of the sham group.