RalGDS induces RalA activation and WPB exocytosis. (A) Activation of GFP-RalA upon coexpression of GFP-RalGDS in HEK293 cells was determined using a Ral-GTP–specific pulldown. Total GFP-RalA levels are shown in the lower panel showing equal expression. (B) HUVECs were transfected with GFP (negative control) or GFP-RalGDS and grown for 48 hours. GFP-expressing cells were incubated with 1 U/mL thrombin (T) or SF medium alone (−) for 45 minutes. WPBs were visualized by immunofluorescent staining of VWF (red). Shown are representative confocal images of HUVECs expressing GFP or GFP-RalGDS. Bars correspond to 10 μm. (C) The numbers of WPBs in individual, randomly selected GFP- and GFP-RalGDS–positive cells were quantified using confocal microscopy. Horizontal bars represent medians. ***P < .001 by Student t test.