Myosin light chain phosphorylation by myosin light-chain kinase determination

Ca²⁺-CaM-dependent phosphorylation of MLC by MLCK was carried out according to the method reported previously.[13] After phosphorylation of MLC₂₀, solid urea and sample solution, which contained bromophenol blue and glycerol, were added to the reaction mixture. Next, 10% glycerol polyacrylamide gel electrophoresis was used to measure the extent of phosphorylation of MLC₂₀. Scion Image Software (Frederick, Maryland, USA), densitometry software from Scion Co., Ltd., was applied to analyze the percentage of phosphorylated MLC₂₀ in the total MLC₂₀. Monophosphorylation of the positive control was chosen as the control and calculated as 100%.