Activation of Rho in BMVECs by monocytes leads to phosphorylation of TJ proteins, and RhoK inhibitor (Y-27632) blocks monocyte migration across the BBB. The content of phosphorylated occludin and total occludin was measured in endothelial cells, endothelial cells cocultured with monocytes, and monocytes cocultured with endothelial cells after pretreatment with the RhoK inhibitor (Y-27632). (A) Representative immunoblots of phosphorylated occludin/claudin-5, total occludin/claudin-5, and internal standard, actin. (B) Ratio of phosphorylated occludin–actin (phosphoserine). (C) Ratio of phosphorylated occludin–actin (phosphotyrosine). (D) Representative immunoblots of phosphorylated claudin-5, total claudin-5, and internal standard, actin. (E) Ratio of phosphorylated claudin-5–actin (phosphoserine). (F) Ratio of phosphorylated claudin-5–actin (phosphotyrosine). Each bar represents mean value ± SEM (n = 4). (G-H) Human brain sections were double immunostained with antibodies to occludin/phosphoserine. While no immunostaining for phosphoserine was found in control brain tissue (G), phosphoserine staining (green) partially overlapped with occludin (red) on brain microvessels in HIVE (H). (G-H) Original magnification, × 400. Images were visualized using a 40 ×/0.75 NA objective and were acquired using a Color View II digital CCD camera. (I) Monocyte migration (105 monocytes applied to BBB constructs) was studied in response to MCP-1 (5 ng/mL in the bottom chamber). Pretreatment of endothelial cells in the top chamber or monocytes (before application to top chamber*) with RhoK inhibitor, Y-27632 (2 hours; 10 μM), diminished monocyte migration across the BBB by 90% and 85%, respectively. Results were normalized to migration of uninfected monocytes without MCP-1 (a value of 100%). Values represent mean of quadruplicate determinations ± SEM.