Fig. 4.

Expression of WT PLM and its Ser⁶⁸ mutants in cultured PLM KO myocytes. Adult cardiac myocytes isolated from congenic PLM KO mice were infected with adenovirus expressing either GFP alone (KO-GFP), GFP + WT dog PLM (KO-PLM), GFP + PLMS68A (KO-S68A), or GFP + PLMS68E (KO-S68E) and cultured for 48 h. Myocytes from WT littermates infected with adenovirus expressing GFP (WT-GFP) and cultured for 48 h were used as controls. Proteins in myocyte lysates (50 μg/lane) were separated by gel electrophoresis and transferred to ImmunBlot polyvinylidene difluoride membranes. Endogenous mouse and WT dog PLM, unphosphorylated and phosphorylated at Ser⁶⁸, was probed with C2 and CP68 anti-PLM antibodies, respectively, whereas dog PLM Ser⁶⁸ mutants were detected with B8 anti-PLM antibody. C2 antibody recognizes predominantly the unphosphorylated COOH-terminus of both dog and rodent PLM, and CP68 antibody is specific for PLM phosphorylated at Ser⁶⁸ (16, 26), whereas B8 antibody recognizes the NH₂-terminus of dog but not rodent PLM (18).