E1841K mutant influences the peripheral distribution and subsequent exocytosis of Rab27a-positive WPBs due to decreased phosphorylation of NMII-A at S1943. (Ai) Western blots of p-S1943 and total NMII-A in HUVECs expressing SCR or shNMII-A that were rescued by flag-tagged NMII-A WT, E1841K and S1943A mutant. (Aii) Ratio of p-S1943 and total NMII-A to loading control α-tubulin of Ai (***P < .001). (B) Immunostaining of VWF (green), flag (white), and Rab27a (red) in HUVECs expressing flag-tagged NMII-A WT, E1841K, and S1943A mutant. The magnified panels showed the peripheral region (a) and the perinuclear region (b). The peripheral region was defined as the region whose distance to nuclear was >10 μm. Scale bars represent 5 μm. (C) Quantitative analysis of the number of Rab27a-positive WPBs in perinuclear and peripheral regions. (D) Quantitative analysis of the number of Rab27a-negative and Rab27a-positive WPBs (n = 16; **P < .01; ***P < .001). (E) VWF multimer distribution of confluent HUVEC medium after 15 minutes of forskolin stimulation. (F) Ratios of high to low molecular weight of VWF multimers in (C) (**P < .01).