Western blot analyses, immunostaining and immunoprecipitation were performed with the following antibodies: mouse cMLCK6 (link), human cMLCK7 (link), phospho-MLC2v (gift from Dr. N. Epstein, NIH)45 (link), MLC2 (ALX-BC-1150-S-L005 Enzo Life Science), α-actinin2 (mouse monoclonal A7811 Sigma, rabbit polyclonal ab68167 Abcam), myomesin (mouse monoclonal, mMAC myomesin B4, Developmental Studies Hybridoma Bank), HA (mouse monoclonal 2367 Cell Signaling, rabbit monoclonal 3724 Cell Signaling); HA-agarose (26182 Pierce); HA-POD (12013819001 Sigma), Myc (mouse monoclonal 2276 Cell Signaling, rabbit monoclonal 2278 Cell Signaling); biotinylated Myc (B7554 Sigma); Myc-agarose (20168 Pierce); Myc-POD (11814150001 Sigma), and GAPDH (MAB374 MilliporeSigma).
Fluorescent staining for the heart sections was imaged using either Leica TCS-SP2 Laser Scanning Confocal Fluorescent Microscope System or an epifluorescent ZEISS Axiovert200M. Fluorescent staining of cardiomyocytes infected with different cMLCK mutants was performed side by side, followed by imaging with the same exposure time below the level of saturation using a ZEISS Axiovert200M. Cell size was measured using fluorescent images of neonatal cardiomyocytes or HA-positive adult cardiomyocytes using ImageJ. Mouse hearts were stained with beta-galactosidase substrate, X-gal, and were photographed as described46 (link).
Fluorescent staining for the heart sections was imaged using either Leica TCS-SP2 Laser Scanning Confocal Fluorescent Microscope System or an epifluorescent ZEISS Axiovert200M. Fluorescent staining of cardiomyocytes infected with different cMLCK mutants was performed side by side, followed by imaging with the same exposure time below the level of saturation using a ZEISS Axiovert200M. Cell size was measured using fluorescent images of neonatal cardiomyocytes or HA-positive adult cardiomyocytes using ImageJ. Mouse hearts were stained with beta-galactosidase substrate, X-gal, and were photographed as described46 (link).
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