WPB proximal myosin motors. (A) Volcano plot of myosin isoforms in close proximity to WPBs, previously identified by Rab27a-targeted APEX2 proximity proteomics. Blue represents significantly enriched in unstimulated cells; green, significantly enriched in PMA stimulated cells; magenta, significantly enriched in HAI stimulated cells; and gray, not statistically significant, compared with mock transfected HUVECs. Paired t test. (B) Unstimulated HUVECs were fixed and subject to IF analysis to localize Myo9B (green) in relation to VWF (blue) and actin (magenta). Myo9B staining was present in the cytoplasm and at the end of actin stress fibers reminiscent of focal adhesions. In some cases, VWF localized proximal to Myo9B puncta. Scale bar, 10 μm. Inset 1 μm (C) western blotting of tubulin and Myo9b in HUVEC lysate after 2 rounds of electroporation of 300 pMoles luciferase (LUC) and Myo9B targeting siRNA. Representative blot. (D) VWF secretion in response to PMA, HAI, and thrombin was assessed by NIR dot blot (n = 3). (E) Western blotting of tubulin and Myo1c in HUVEC lysate after 2 rounds of electroporation of 500 pM LUC and Myo1c targeting siRNA. (F-G) LUC and Myo1c KD HUVEC were exposed to PMA (100 ng/mL) or (G) VEGF165 (40 ng/mL), and VWF secretion was quantified by NIR dot blot (n = 3). t test. ∗∗∗P < .005; ∗∗P < .01. (H-I) Western blotting and (I) densitometry of Myo1c, VEGFR2, and GAPDH in LUC and Myo1c KD HUVEC (n = 6). (J-K) HUVECs were treated with the pan class I myosin inhibitor PCLP for 16 hours and endogenous levels of GAPDH and VEGRF2 determined by western blotting. One-way analysis of variance (ANOVA), ∗P < .05 (n = 3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KD, knock down; NIR, near infrared.