MCAO induces occludin S490 phosphorylation in a tPA-dependent manner. Mice were subjected to MCAO and killed at 3 and 24 hours after MCAO. (A) Stitched images of the region adjacent to the ischemic core (Isch) showing vessel pS490 occludin (red) and total occludin (green) staining in WT mice compared with contralateral hemisphere. Scale bar, 60 µm. (B) Confocal images zoomed in on individual vessels showing pS490 occludin (red) and ZO-1 (white) staining in tPA^−/− mice after MCAO compared with WT mice 24 hours’ post-MCAO. Scale bar, 17 µm. (C) Quantification of pS490 fluorescence intensity at the junction 24 hours after MCAO in WT and tPA^−/− mice in the contralateral (Contra) and ipsilateral (Ipsi) regions. (D) The amount of pS490 and total occludin in extracts from the ipsilateral (I) and contralateral (C) hemispheres was determined by western blot in WT and tPA^−/− mice at 3 and 24 hours’ post-MCAO. (E) Quantification is expressed as relative to WT contralateral from each time point. (F) ICV injection with either PBS or tPA was performed in WT mice; after 6 hours, protein levels of pS490 and total occludin were determined by western blot of the whole brain extract. (G) Quantification is shown. One-way analysis of variance followed by Holm-Šídák post hoc test was used for comparison of ≥3 groups; a t test was used for comparison between 2 groups. *P < .05, ***P < .001, ****P < .0001. ns, nonsignificant; Veh, vehicle.