Fig. 2. Changes in the dephosphorylating activity against ³²P-labeled MARCKS during myogenic differentiation. (A) Soluble extracts were prepared from myoblasts that had been cultured for the indicated periods, and aliquots of them (30 µg/lane) were incubated with ³²P-labeled MARCKS (0.3 µg) for 30 minutes at 30°C. The extracts from the 72 hour cultured cells were also incubated as above but in the presence of 100 nM okadaic acid (lane 72O) or 5 nM tautomycin (lane 72T). As a control (lane C), ³²P-labeled MARCKS was incubated as above but without any addition. The samples were then subjected to SDS-PAGE followed by autoradiography. Note that the rate of dephosphorylation reaction was linear up to 30 minutes when incubated with the extracts of the cells cultured for 48 hours in the absence of okadaic acid. The arrows indicate ³²P-labeled MARCKS. (B) The same extracts were subjected to immunoblot analysis using an anti-phospho-MARCKS antibody. The arrowhead shows phospho-MARCKS. (C) The cells were cultured in the absence () and presence of 25 nM okadaic acid () or 0.5 µM tautomycin ([UNK]) for the indicated period, and the degree of cell fusion was determined as in Fig. 1.