Anti mouse rhoa

U87 cells were trypsinized, then plated onto 6-well culture plates with suitable culture medium (high-glucose DMEM, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin) containing 10³ cells. When 80% of the U87 cells were confluent, the complete culture media was aspirated and replaced with fresh EBM-2 medium containing 5% fetal bovine serum. EC monolayers grown on glass coverslips were placed into culture plates and co-cultured for 4 days. The GEC monolayers grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking with 2% bovine serum albumin (BSA) in PBS, the cells were incubated with mouse anti-occludin (diluted 1:50; Life Technologies Corporation, Frederick, MD, USA) and rabbit anti-claudin-5 (diluted 1:50; Life Technologies Corporation, Frederick, MD, USA) to visualize the distribution of occludin and claudin-5, respectively. The cells were incubated with rhodamine-labeled phalloidin (diluted 1:250; Sigma-Aldrich) to assess actin filaments. The cells were incubated with mouse anti-RhoA (diluted 1:150; Abcam, Cambridge, UK) and rabbit anti-ROCK II (diluted 1:50; GeneTex, Irvine, California, USA) to analyze their expression. The glass slides were then evaluated using immunofluorescence microscopy (Olympus, Japan).