Fig. 5
MYPT1 promotes dephosphorylation of MLC2 in endothelial cells. a Immunoblot analysis of HEK-293 cells treated with vehicle or GDC-0326 for 48 h using the indicated antibodies. Endogenous MYPT1 was immunoprecipitated and its ability to interact with actin was assessed in an overlay assay. Bars to the right show quantification of actin bound to total MYPT1 from three independent experiments. b Western blot analysis of MYPT1, pS20 MLC2 and β-actin in lysates of wild-type mouse lung endothelial cells transfected with siControl (siCtrl) or siMYPT1. Bars to the right show quantification of pS20 MLC2 normalized to β-actin from three independent experiments. c Images of endothelial cells transfected with siCtrl or siMYPT1, seeded on gelatin-coated plates 72 h post-transfection, and immunostained for β-catenin (green), pS20 MLC2 (red) and F-actin (blue). d Quantification of total cell pS20 MLC2 immunostaining intensity (shown as integrated density) of images shown in c (n ≥ 6 independent experiments). Scale bars, 15 µm (c). Data in a, b, and d represent mean ± SEM (error bars). *P < 0.05, **P < 0.01. Statistical analysis was performed in a, and b by the two-sided Student’s t test and in d by the two-sided Mann–Whitney test
