Rab27A, Rab3B, and Rab3D recruitment to WPBs is decreased upon MADD silencing. (A-C) HUVECs were transduced with pLKO-shRNAs targeting Rab27A (A), Rab3B (B), and Rab3D (C) or nontargeting control (shCTRL); representative western blots and quantifications of 3 to 5 biological replicates are shown confirming knockdown after 7 days. Data are shown as mean ± SEM and were analyzed by using an unpaired Student t test. Dotted lines indicate activity levels in shCTRL. ***P < .001; ****P < .0001. (D,F) HUVECs were transduced with a pLKO-GFP-shRNA co-expression construct containing shCTRL, shRab27A, shRab3D, or shMADD (all shown in green) and immunostained for VWF (blue) and Rab27A or Rab3D (red) as indicated. Individual channels are shown in grayscale below. Boxed areas are magnified on the right. Yellow arrowheads indicate Rab⁺ and cyan arrowheads indicate Rab^– WPBs. Scale bars represent 10 µm. (E,G). The Rab⁺ proportion of WPBs per cell was quantified. (E) The proportion of Rab27A⁺ WPBs was divided over 3 bins based on the amount of WPBs in shCTRL (n = 56) and was compared with shRab27A (n = 55) and shMADD (n = 54). Data are shown as mean ± standard deviation (SD) and were analyzed by using two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. ****P < .0001. (G) The proportion of Rab3D⁺ WPBs in shCTRL (n = 42) is compared with shRab3D (n = 31) and shMADD (n = 33) without binning. Data are shown as mean ± SD and were analyzed by using one-way ANOVA with Tukey’s multiple comparisons test. ****P < .0001. ns, not significant.