Figure 7 Autophosphorylation activity of different PKC isoforms. Quiescent cells were restimulated for 7 h with FCS in the absence (C) or presence of either 50 |iM a-tocopherol (a) or ß-tocopherol (ß). PMA (100 nM) was added for the last hour of the preincubation period. Then, cell extracts were prepared, and PKC isoforms were immunoprecipitated with the indicated antibodies. Autophosphorylation reaction of the individual isoforms was performed as described in Methods. Samples were electropho-resed and blotted, and the calculated ratio between incorporated radioactivity [52P] and the protein levels (Western blot) is represented in the bar graphs for each condition. Data are representative of three independent experiments.
FCS for 7 h in the presence or absence of a-tocopherol, PKCa was immunoprecipitated, blotted, and the 32P incorporation measured. Figure 8 shows a bar graph presentation of the radioactivity intensities integrated and normalized with respect to the protein levels of each sample. Relative to the control (N), the PMA-treated cells (P) showed a significant increase in 32P incorporation into PKCa. Cells pretreated with a-tocopherol (column a) showed a large inhibition of PKCa phosphorylation, whereas cells preincubated with P-tocopherol (column P) showed much less inhibition. The inhibitory effect of a-tocopherol was reversed by two potent protein phosphatase inhibitors, oka-daic acid 2 nM (not shown) or calyculin A 2 nM (column a + C). Figure 9 shows the PKCa activity measured after immunoprecipitation of the enzyme
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