GFP-RalGDSΔ382-597 blocks stimulus-induced WPB exocytosis. (A) Schematic representation of GFP-tagged RalGDS and RalGDSΔ382-597, which lacks the catalytic CDC25 domain. (B) RalA activation upon coexpression of GFP-RalGDS or GFP-RalGDSΔ382-597 with GFP-RalA was determined using a Ral-GTP–specific pulldown. Middle and bottom panels show total GFP-RalA, and GFP-RalGDS and GFP-RalGDSΔ382-597, respectively, to confirm equal expression. A vertical line has been inserted to indicate a repositioned gel lane. (C) HUVECs were transfected with GFP (negative control) or GFP-RalGDSΔ382-597 and grown for 48 hours. Cells were incubated with 1 U/mL thrombin, 10 μM epinephrine, and 100 μM IBMX or SF medium alone (unstimulated) for 45 minutes. WPB numbers in GFP- and GFP-RalGDSΔ382-597–positive cells were quantified using confocal microscopy. Horizontal bars represent medians. ***P < .001 by Student t test.