siRNA-mediated knockdown of MyRIP increases stimulus-induced exocytosis of VWF and results in fewer peripheral WPBs. (A) HUVECs were treated in 2 consecutive rounds with 2 different siRNA oligonucleotides (at both 100 and 200 pmol) directed against MyRIP or a control oligonucleotide (at the highest oligonucleotide concentration 200 pmol). Western blot analysis 48 hours after the second round showed down-regulation of MyRIP using a goat anti-MyRIP antibody, whereas tubulin levels were unaffected. (B) Samples of media were acquired after 30 minutes with and without 100 ng/mL PMA, and then cells were lysed and the VWF levels of each sample quantified by ELISA. Basal and stimulated release was normalized to total VWF. (C,E) Mock and (D,F) MyRIP (oligo 1, 200 pmol) and control siRNA-treated cells were plated out subconfluently and visualized by immunofluorescent staining. (C,D) Confocal images were acquired of MyRIP (red), VWF (blue), and actin (green). (E,F) A 40-μm circle was placed at the cell nucleus, and the percentage of VWF-positive structures (white) present inside and outside the circle was determined (“Quantification of WPB distribution” in “Methods”). (F) Scale bar represents 10 μm. (G) Eighteen to 20 cells were randomly selected from each condition from 3 separate experiments. The percentage present inside the 40-μm circle was plotted. ***P < .001 by Student t test and χ² test.(B,G) Error bars represent SD.