Fig. 5. Transient overexpression and subcellular localization of MARCKS and its mutant forms in cultured myoblasts. Myoblasts that had been cultured for 24 hours were transfected with the cDNAs for MARCKS (Wt) and its mutant forms (N/S and A₂G₂) and further incubated for 48 hours in the absence (a) or presence of 100 nM okadaic acid (b). After incubation, total cell lysates were prepared and subjected to immunoblot analysis using anti-MARCKS (A) or anti-Myc antibody (B). Lanes C represent the mock transfected with empty vector. Total cell lysates were also separated into soluble and membranous fractions, which were then subjected to immunoblot analysis using anti-Myc antibody (C). Letters S and P represent the supernatant (soluble) and pelleted (membrane) fractions, respectively. The arrowhead indicates the endogenous MARCKS, and the arrows show the Myc-tagged MARCKS proteins.