Fig. 3. Inhibition of immunoprecipitated kinase activity by a PKC-specific inhibitor. (A) PKC was immunoprecipitated from myoblasts with or without PMA treatment. After immunoprecipitation with an antibody specific for PKC, in vitro kinase assays were carried out in the presence and absence of the PKC inhibitor, chelerythrine (2 µM), using either histone or MBP as a substrate. The phospho-proteins were loaded on a 10% or 12% SDS-polyacrylamide gel then transferred to nitrocellulose followed by autoradiography to assess histone phosphorylation (upper row) or MBP phosphorylation (middle row). The blots were probed with an anti-PKC antibody to confirm equal amounts of PKC protein in each sample (lower row). (B) The incorporation of ³²P into histone or MBP from experiments such as that shown in A was quantified. These results presented are averaged from two separate experiments and demonstrate the marked induction of PKC activity by PMA that is maintained after immunoprecipitation and inhibited by chelerythrine.

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