FIGURE 3.

ROCK and PRK are both required for thrombin-induced permeability. a and b, permeability assays were done in 3-day-old human endothelial cell starved overnight and pre-treated with C3 toxin (1 μg/ml) with tetanolysin (20 units) for 16 h, Y27632 (45 min, 5, 10, and 50 nm), blebbistatin (45 min, 5 μm), and ML-7 (45 min, 10 μm) prior VEGF and thrombin stimulation as described previously. c, human endothelial cells were electroporated with mock or Myc-tagged ROCK-KD mutant and cultured for 3 days on collagen-coated inserts. Overnight starved cells were then stimulated with thrombin and further analyzed for permeability. Expression level of ROCK-KD was checked by Western blot against Myc. d, similarly, cells were transfected with nonsilencing RNA (nsi, 3 days, 50 nm) or two independent sequences targeting PRK (si#1 and si#2, 3 days, 50 nm). Extinction efficiency was checked by Western blot for PRK, and tubulin was used as a loading control. All panels are representative of at least three independent experiments. Permeability assays were performed in each condition in nonstimulated (Ctrl), and VEGF and thrombin-stimulated cells. ANOVA test: *, p < 0.05; **, p < 0.01; ***, p < 0.001.