CSF-1 activates Raf-1 but not B-Raf or A-Raf and none is activated in the presence of cAMP and CSF-1. (A) Western blot analysis of the three Raf isoforms in 32D/CSF-1R cells. Where indicated, blocking peptide (1 and 3 μg/mL) was preincubated with the appropriate antibody. The middle panel is a longer exposure of the B-Raf segment of the blot shown on the left. Equal amounts of lysates (50 μg) from 32D/CSF-1R and PC12 cells were also Western blotted for B-Raf, showing significantly higher levels of expression in PC12 cells (right panel). “+” refers to a parallel blot that was incubated with anti–B-Raf and its blocking peptide (1 μg/mL). (B) 32D/CSF-1R cells were treated as described in Fig 2 and Raf-1 or B-Raf was immunoprecipitated. Kinase activity was measured using a recombinant KD-MEK1 as substrate. Shown is a plot summarizing data (means ± SE) from multiple (n) experiments. Only CSF-1–stimulated Raf-1 activity is significant (*) compared with basal (P < .05).