FIG. 1.

B-Raf and Raf-1 form stimulation-inducible heterodimers. (A) Coimmunoprecipitation of overexpressed Raf proteins. COS-1 cells were transiently transfected with Flag-tagged Raf-1 and HA-tagged B-Raf. Cells were starved overnight and stimulated with TPA for 10 min. Lysates were immunoprecipitated with the Flag antibody and probed for associated B-Raf or immunoprecipitated with the HA antibody and probed for associated Raf-1. (B) Endogenous heterodimers are formed in COS-1 cells. Starved COS-1 cells were stimulated with 100-ng/ml TPA for 10 min or EGF for 5 min. B-Raf/Raf-1 heterodimers were detected by immunoprecipitation with a B-Raf antibody, followed by immunoblotting for Raf-1 and vice versa.