FIGURE 10.

Expression of GEF-H1 phospho-deficient mutant abolished protective effects of ANP in the thrombin model of EC barrier dysfunction. HPAECs transfected with the wild type GEF-H1 or the GEF-H1-S885A mutant were stimulated with ANP (100 nm, 20 min) prior to thrombin (0.5 units/ml) challenge. A, transendothelial resistance reflecting EC monolayer barrier properties was monitored over 2 h in transfected ECs stimulated with thrombin or ANP + thrombin. Data are expressed as mean ± S.D. Inset, expression of GFP-GEF-H1 wild type or GFP-GEF-H1-S885A in HPAECs. B, MLC phosphorylation in cell lysates was analyzed by Western blot with phosphospecific antibodies. Efficiency of siRNA-induced PAK1 depletion and equal protein loading were confirmed by membrane reprobing with PAK1 and total β-tubulin antibodies, respectively. *, p < 0.05 versus thrombin alone. Error bars, S.D.