Fig. 8.

APC-induced cytoprotective signaling is mediated by Dvl-2. (A and B) Endothelial cells transfected with 50 nM nonspecific (ns) or Dvl-2 siRNAs were incubated with 10 nM thrombin (Th) or 20 nM APC for 5 or 180 min at 37 °C. Cells were lysed and incubated with GST-PBD or GST-RBD, and the amount of activated Rac1 or RhoA was detected by immunoblotting. Total cell lysates were immunoblotted with anti-Rac1 or anti-RhoA antibody as a control. Inset shows an anti–Dvl-2 and anti-actin antibody immunoblot of total cell lysates. The data (mean ± SEM) are expressed as the fold increase over untreated control (Ctrl) from three independent experiments. The differences between Rac1 or RhoA activation observed in agonist-treated cells versus untreated control cells were significant (*P < 0.05; **P < 0.01). (C) Serum-deprived endothelial cells (EC) transfected with 50 nM nonspecific or Dvl-2–specific siRNAs were preincubated with or without 20 nM APC for 3 h at 37 °C. Inset shows a total cell lysate immunoblot using the anti–Dvl-2 and anti-actin antibodies. Cells then were stimulated with 10 nM Th or 20 nM APC for 10 min at 37 °C, and endothelial barrier permeability was assessed. Data (mean ± SEM) are expressed as the fraction of thrombin-induced endothelial barrier permeability measured at 10 min and are representative of three independent experiments. The differences in thrombin-induced endothelial barrier permeability in cells transfected with nonspecific siRNA versus cells transfected with Dvl-2 siRNA was significant (**P < 0.01).