FIGURE 3.

Impaired epinephrine-stimulated Rap1 activation in endothelial cells after down-regulation of Epac1. HUVECs treated with Epac1 siRNA (siEpac1) or control siRNA (siCTRL) were preincubated with SF medium for 1 h. Subsequently, cells were stimulated with 10 μm epinephrine and 100 μm IBMX (Epi) or 1 unit/ml thrombin (Thr) for the indicated minutes. Activation of Rap1 in HUVECs was determined using a Rap1-GTP specific pulldown. Western blots of Rap1-GTP illustrate the inability of Epac1 down-regulated endothelial cells to activate Rap1 in response to epinephrine, although thrombin-induced Rap1 activation remains unaffected. The total amount of Rap1 shown in the middle panel is used as loading control.