Fig. 2. PKC isozyme expression and activation in muscle cells. (A) PKC isozyme expression was determined by western blot analysis in mouse myoblasts. Myoblasts were grown for 2 days then lysed in RIPA buffer. 80 µg of proteins were chromatographed on a 7.5% SDS gel then probed with antibodies specific for individual PKC isozymes. The lanes containing protein from myoblast cultures are labeled `Mb'. Adjacent lanes include positive controls for each isozyme. `B' refers to mouse brain extract (20 µg/lane), which is known to highly express all the isozymes except for PKC and was therefore used as a positive control. For PKC, extract of skeletal muscle tissue [`Mf' (myofibers)] was used (80 µg/lane). (B) PKC isozyme activity increases upon integrin binding and activation. PKC isozymes were immunoprecipitated from total cell extracts of myoblasts plated on FN for different lengths of time, and the activity of each isozyme was determined by the level of ³²P incorporation into histone III-S. These results are normalized to the activity at time zero and to the amount of PKC isozymes immunoprecipitated from each sample determined by western blot analysis. These results represent the mean±s.d. from eight separate experiments. (C) 5-expressing and 5-deficient myoblasts were plated on FN for various times. PKC isozymes were immunoprecipitated as described in A, and phosphorylated histone was analyzed on 10% SDS-polyacrylamide gel. A representative autoradiogram is shown. As in A, PKC isozyme activity increases transiently in 5-expressing myoblasts (5⁽⁺⁾) plated on FN. By contrast, there was no increase in PKC isozyme activity in the 5-deficient myoblasts (5^-/-) on FN. The protein levels of each isozyme in the two cell populations were indistinguishable by western blot analysis.

Return to article