Interaction between myosin IIa and zyxin is required for cAMP-mediated actin framework formation and WPB exocytosis. (A) Silver-stained gels showing immunoprecipitates from HUVECs with or without forskolin stimulation. Peptide sequences derived from the myosin IIa band (MyoIIa) were obtained by mass spectrometry. (B) Immunoprecipitation between myosin IIa and zyxin using HUVECs (bottom) or zyxin-flag-overexpressing HUVECs (upper). The immunoprecipitates were immunoblotted with either zyxin antibody (zyxin) or myosin IIa antibody (MyoIIa). (C) Immunoprecipitation of myosin IIa with the indicated antibodies using lysates of HUVECs expressing WT or the zyxin, zyxin Δ2-42, zyxin Δ2-137, or 4FA mutant with forskolin stimulation. (D) HUVECs coexpressing mCherry-PSL-lum (PSL-lum, magenta) and Lifeact-GFP (F-actin, green) were infected with shRNAs against zyxin (shZyxin), followed by introduction of WT or the zyxin Δ2-42 mutant. Images show the basal state (0 min) and the last imaging points (15 min; n = 4; 2 independent experiments). (E) Average numbers of exocytotic events in cells used in D (n = 4; **P < .01; 2 independent experiments; scale bars, 5 μm in A, B, and E, 1 μm in insets; mean ± standard deviation; Student’s t-test).