Figure 5.

Rho-dependent mechanism of KRIT1-mediated barrier preservation of ECs exposed to 18% CS and TRAP6. ECs grown on Bioflex plates and transfected with nonspecific or KRIT1-specific siRNA were subjected to 18% CS for 2 hours followed by TRAP6 (50 ng/ml, 15 min) stimulation with or without PC (200 ng/ml) cotreatment. (A) Rho activation was evaluated by RhoGTP pulldown assay and normalized to total Rho content in cell lysates. (B) Activation of Rho pathway was evaluated by Western blot analysis of phospho-myosin light chain phosphatase (pMYPT1) and diphospho–myosin light chain (ppMLC) levels. Reprobing with β-actin antibody was used as a normalization control. KRIT1 knockdown was verified by Western blot and represented approximately 50% of the original KRIT1 level. (C) ECs were transiently transfected with plasmids encoding wild-type KRIT1 and KRIT1-R452E mutant, and activation of Rho pathway was evaluated by Western blot analysis of ppMLC levels. Bar graphs depict results of membrane densitometry analysis. Data are expressed as mean ± SD. *P < 0.05 (n = 5). RDU, relative density units.