(A) A putative 20-kDa substrate that was labeled with P³² in the presence of cardiac-MLCK was identified in fractionated murine myocardium extracts (arrows). Fraction numbers are shown at top. (B) P³²-labeled MLC2v was purified and visualized by autoradiography (left lane) and silver staining (right lane). (C) Peptides from the purified protein, which matched the sequences of murine MLC2v, are shown in red. (D) Purified MLC2v from murine myocardia was phosphorylated by cardiac-MLCK in a Ca²⁺-calmodulin–dependent manner. (E) RcMK detected rat cardiac-MLCK from cultured cardiomyocyte cell extracts and FLAG-tagged murine cardiac-MLCK. (F) Nonphosphorylated MLC2v and phosphorylated MLC2v were separated using urea-glycerol gel electrophoresis. tMLC and p-s15MLC were confirmed to specifically detect each target protein. (G) Overexpression of murine cardiac-MLCK in cultured cardiomyocytes following infection with an adenovirus vector encoding murine cardiac-MLCK at MOIs of 90 and 150 upregulated the phosphorylation of MLC2v in a dose-dependent manner. Endogenous rat cardiac-MLCK is shown at top; overexpressed murine cardiac-MLCK is shown below. (H and I) Both si-cMK-1 and si-cMK-3 effectively suppressed the mRNA (H) and protein levels (I) of cardiac-MLCK, resulting in reduced phosphorylation of MLC2v. smMLCK, α-actinin, desmin, and troponin T were not affected by suppression of cardiac-MLCK expression. siCTL, control siRNA. (J) The protein levels of smMLCK were effectively decreased by si-smMK; no remarkable changes were observed in protein levels of phosphorylated MLC2v or other sarcomere-related proteins.