Protein S mediates Rac1-dependent barrier protection and activates S1P₁ after Tyro3 ligation in OGD-treated human BBB monolayers. (A) Time course of Rac1 activation (Rac1-GTP) in human BBB monolayers in vitro after OGD (0-60 minutes) with and without PS (100nM). Graph: relative abundance of Rac1-GTP signal intensity normalized by Rac1 (0-120 minutes). P < .01, OGD + PS versus OGD − PS. (B-C) Permeability to FITC-dextran (B) and TER (C) in BBB monolayers 2 hours after OGD and treatment with PS (100nM) after transduction with Ad.Rac1 and Ad.GFP, transfection with S1P₁ siRNA and control siRNA, and incubation with S1P₁-specific antagonist W146 (50nM). (D) Immunoprecipitation (IP) of S1P₁ with anti-S1P₁ antibody followed by immunoblotting (IB) with antiphosphothreonine antibody (pThr) in BBB monolayers 10 to 60 minutes after OGD and treatment with PS with and without anti-Tyro3 (N-terminus) antibody or nonimmune IgG. (E) IP of S1P₁ with anti-Tyro3 (C-terminus) and control nonimmune IgG in BBB monolayers 10 minutes after OGD and treatment with PS (100nM) with and without control antibody or anti-Tyro3 (N-terminus) ligand-binding domain–blocking antibody. (F) IP of Tyro3 with anti-S1P₁ antibody and control nonimmune IgG in BBB monolayers 10 minutes after OGD and PS (100nM) treatment with and without control antibody or anti-Tyro3 (N-terminus) blocking antibody. (G) Akt phosphorylation (pAkt, Ser473) in BBB monolayers 10 to 60 minutes after OGD with or without PS and with and without Tyro3 (N-terminus) blocking antibody. Graph: relative abundance of pAkt signal intensity normalized by total Akt. (H) IP of S1P₁ with anti-S1P₁ antibody followed by IB with antiphosphothreonine or anti-S1P₁ antibodies in BBB monolayers pretreated for 1 hour with vehicle or LY294002 (10μM) followed by OGD and PS treatment for 10 minutes. In all studies, human PS was used at 100nM. Mean ± SEM, from 3 to 6 independent cultures.