FIG. 8.

CaM inhibitors prevent the acute activation of Raf kinases after NGF stimulation. PC12 cells were pretreated (+) or not pretreated (−) for 1 h with W12 or W13 and stimulated (+) or not stimulated (−) for 5 or 45 min with NGF. After treatment, cells were lysed and protein extracts were subjected to immunoprecipitation with specific antibodies against Raf-1 (A), B-Raf (B), or A-Raf (C). Immunoprecipitates were used to determine kinase activity with wild-type MEK1 as a substrate (upper panels) and analyzed by Western blotting with the same antibody used in the immunoprecipitation step as a control for the enzyme content in the immunoprecipitates (lower panels). Graphs in panels A and B show the average Raf activity (expressed as fold induction over the kinase activity obtained in untreated cultures) from three independent experiments. ∗∗, P value of <0.01, as determined by Student's t test. A-Raf activity was not quantifiable in any of the experimental conditions. IgG, immunoglobulin G.