RalGDS knockdown by siRalGDS impairs stimulus-induced exocytosis of WPBs. (A) HUVECs were treated in 2 consecutive transfection rounds with a pool of 4 siRNA oligonucleotides directed against RalGDS (siRalGDS) or a control siRNA oligonucleotide (siCTRL). Western blot analysis 48 hours after transfection showed down-regulation of RalGDS expression, while α-tubulin and RalA remained unaffected. (B) Activation of RalA in siCTRL- and siRalGDS-treated HUVECs in response to thrombin was determined using a Ral-GTP specific pulldown. Cells were preincubated with SF medium for 1 hour, after which they were stimulated for 2 minutes with 1 U/mL thrombin (T₂) or SF medium alone (−). All lysates contained similar amounts of RalA (bottom panel). (C) Representative confocal images of siCTRL- and siRalGDS-treated HUVECs that were incubated for 45 minutes with 1 U/mL thrombin, 10 μM epinephrine, and 100 μM IBMX or SF medium alone (unstimulated). WPBs were visualized by immunofluorescent staining of VWF (green), while staining of β-catenin (red) was used to delineate the cell membrane. Bars correspond to 10 μm. (D) Numbers of WPBs in individual cells were quantified as described in “CaM pull-down assays.” Approximately 15 randomly selected cells were counted for each experimental condition. Shown is the analysis of 1 experiment representative of 3 independent experiments. Horizontal bars represent medians. ***P < .001 by Student t test.