Loss of CD63 does not affect WPB formation, exocytosis or maturation. HUVECs were treated for 2 rounds with 2 different siRNA oligonucleotides, individually or pooled, directed against CD63 or a control oligonucleotide. (A) Representative maximum intensity projection confocal images of immunofluorescence staining of CD63 and VWF and P-selectin in control and CD63 pooled siRNA treated cells showing depletion of CD63. Merged panel shows CD63 in red, VWF in blue, and P-selectin in green. Cell morphology, WPB formation, and P-selectin recruitment to WPBs remain unaffected by transfection of siRNA. Scale bars represent 25 μm. (B) Western blot analysis of cell lysates for control and 2 separate or pooled CD63 targeted siRNA treated cells showing down-regulation of CD63 using mouse–anti-CD63, with β-tubulin as a loading control. (C) Representative secretion assay data for mock-transfected and CD63 depleted cells, where cells were incubated with serum free release media (basal) or release media containing PMA (100 ng/mL; stimulated) for 45 minutes. Results represent percent total VWF secreted in basal and stimulated conditions. Data are shown as mean ± SEM (n = 3).