Fig. 4. Raf kinase activities. (A) Time course of activation of immunoprecipitated A-Raf, B-Raf and Raf-1 in the kinase cascade assay in raf-1+/+ cells following stimulation with EGF. The conditions for the assay were the same as those used for routinely measuring B-Raf activity. (B) Time course showing the fold Raf-1 activation in raf-1+/+, raf-1–/– and raf-1FF/FF MEFs following EGF stimulation. Standard conditions for measuring Raf-1 activity were utilized. (C) Time course showing the fold A-Raf activation in MEFs following EGF stimulation. The conditions were the same as for the Raf-1 assay. (D) Time course showing the fold B-Raf activation in MEFs following EGF stimulation. The B-Raf assay conditions were utilized. Each experiment was performed in triplicate and error bars show standard deviations.