Phosphorylation of different myosin IIa sites plays distinct roles in regulation of VWF secretion. (Ai) Western blot of phospho-myoIIa at S1916 (ppS1916) and S1943 (ppS1943) in HUVECs stimulated with forskolin for different points. (Aii) Ratio of phosphorylated myoIIa at S1916 (p1916) and S943 (p1943) to total myoIIa of left. (Bi) Western-blot of myosin mutants expressed in HUVECs. (Bii) Enzyme-linked immunosorbent assay of VWF secretion from HUVECs expressing SCR and shMyoIIa in the presence of forskolin, rescued by WT myosin IIa (WT) and the S1916A, S1943A, or S1916/1943A mutants of myosin IIa (n = 4; *P < .05, **P < .01, 3 independent experiments). (C) Immunostaining of VWF (yellow), F-actin (cyan), and paxillin (magenta) in HUVECs used in B. (D) Percentage of WPBs along the FA-anchored stress fibers in C. (Ei) Western blot of phospho-myoIIa at S1916 (p1916) and S1943 (p1943) in SCR and shZyxin HUVECs stimulated with forskolin for 15 minutes. (Eii) Ratio of p1916 and p1943 to total myoIIa of left. (Fi) Western blot of p1916 and p1943 in SCR HUVECs and shCK2 HUVECs stimulated with forskolin for 15 minutes. (Fii) Ratio of p1916 and p1943 to total myoIIa of left. (Scale bars, 5 μm in C; mean ± standard deviation; NS > 0.05, *P < .05, **P < .01, ***P < .001, Student’s t-test.)