Protein S protects the endothelial barrier integrity in OGD-treated human BBB monolayers and mouse brain endothelial cells via Tyro3. (A) Tyro3 and Axl immunoblotting in human endothelial monolayers transfected with Tyro3 or Axl siRNAs. (B-C) Permeability (P) to FITC-dextran (B) and transmonolayer electrical resistance (TER; C) in human BBB monolayers transfected with Tyro3 or Axl siRNAs or incubated with Tyro3, Axl, and Mer N-terminus–specific blocking antibodies 2 hours after OGD with and without human PS (100nM). (D) Human PS dose-dependently phosphorylates Tyro3 in human BBB monolayers 15 minutes after OGD. IP indicates immunoprecipitation of phosphotyrosine proteins; IB, immunoblotting of Tyro3. Cells were treated with PS and anti-Tyro3 (C-terminus) or anti-Tyro3 (N-terminus) antibody. P < .05 versus vehicle. (E) Confocal microscopy analysis of Tyro3 distribution in a human BBB monolayer. Top (x-y axis): an image of compressed Z-stacks. Tyro3, green; nuclei, blue. Bottom (x-z axis): Z-stacks image of Tyro3 (green) at the apical and basolateral side. (F) Confocal microscopy analysis of S1P₁ distribution in a human BBB monolayer. Z-stacks image (x-z axis) of S1P₁ (red) at the apical and basolateral side. (G) Survival of subconfluent mouse brain endothelial cells derived from Tyro3, Axl, and Mer mutants and littermate controls 24 hours after OGD with and without mouse PS (50nM). wt indicates wild-type mice. Mean ± SEM, from 3-6 independent cultures.