Motor and rod domain mutations associated with Myh9-RD differentially affect cell adhesion and NMIIA functions. (A-F) Vinculin staining of MKs spread on collagen-1 coverslips. Scale bar, 25μm. Images were acquired with a Plan-Fluor ×63 objective (N.A. 1.4) of a Zeiss LSM 780 confocal microscope with Zeiss AxioCam. Images were processed with ZEN software available from Zeiss. Analysis of vinculin-positive focal adhesion sites were performed with ImageJ. (G) Scatterplots representing median and interquartile range of the distribution for areas of vinculin-positive structures in WT and Myh9-RD mice MKs. (H) Scatterplots representing median and interquartile range of the distribution for vinculin-positive sites per unit area in MKs from Myh9-RD and WT littermate control mice (WT^GFP+/−, n = 25; R702C^GFP+/−, n = 25; WT, n = 20; D1424N^+/−, n = 20; WT, n = 15; E1841K^+/−, n = 15). (I) Representative immunoblots showing phosphorylated RLC (RLC^Ser19) and total RLC levels or showing phosphorylated myosin HC (NMIIA^Ser1943) and total NMIIA-HC levels in WT and mutant MKs. (J) Graph represents quantification of immunoblots as mean ± standard error of the mean from ≥3 experiments