FIG. 9.

Effect of 8-pCPT-cGMP on EGF-induced c-Raf kinase activation measured by a coupled enzymatic assay. Wild-type BHK (A) and clone C11 (B) cells were treated as described in the legend to Fig. 5, and c-Raf kinase was immunoprecipitated as described in Materials and Methods. Washed immunoprecipitates were sequentially incubated with recombinant, nonactivated MEK and MAP kinase in the presence of nonradioactive ATP; for the last 10 min of the reaction, MBP and [γ-³²PO₄]ATP were added, and ³²PO₄ incorporation into MBP was assessed as described for Fig. 5. The middle panel is a Western blot, probed for c-Raf kinase, of the gel shown in the top panel and demonstrates equal amounts of c-Raf kinase present in the immunoprecipitates. The values in the lower panel represent the means ± SD of three independent experiments.