Fig. 3. Identification of protein phosphatase responsible for dephosphorylation of MARCKS in cultured myoblasts. (A) Effects of increasing concentrations of tautomycin and okadaic acid on dephosphorylation of ³²P-labeled MARCKS. Soluble extracts were prepared from the cells cultured for 48 hours and subjected to the phosphatase assay in the presence of increasing concentrations of tautomycin () and okadaic acid ([UNK]) as described in Materials and Methods. The resulting samples were subjected to SDS-PAGE followed by autoradiography. The bands corresponding to ³²P-labeled MARCKS were quantified using a densitometer. The intensity of the band seen without any treatment was expressed as 100% and the others were expressed as relative values. Each of the data points represents mean±s.e. of triplicate determinations. (B) Effect of immunoprecipitation with anti-PP-1C IgG on dephosphorylation of ³²P-labeled MARCKS. Soluble extracts were prepared from the cells cultured for 48 hours and incubated with 10 µg of preimmune IgG (lane b) or anti-PP-1C IgG (lane c) at 4°C for 2 hours. After incubation, the samples were treated with protein A-Sepharose and centrifuged. The resulting supernatants were then subjected to the phosphatase activity assay followed by SDS-PAGE and autoradiography. Lane a represents ³²P-labeled MARCKS incubated alone. (C) Effects of protein phosphatase inhibitors on dephosphorylation of ³²P-labeled MARCKS. Soluble extracts were prepared and assayed for their phosphatase activity as above but in the absence (lane a) or presence of 20 nM protein phosphatase inhibitor-2 (lane b), 100 nM protein phosphatase-2A inhibitor (lane c), or both (lane d). Lane e represents ³²P-labeled MARCKS incubated alone. The arrows indicate the phosphorylated MARCKS.