FIG. 4.

14-3-3 proteins enhance Raf heterodimerization. (A) COS cells were cotransfected with Flag-Raf-1, HA-B-Raf, and increasing amounts of GST-14-3-3. Lysates from serum-starved cells were immunoprecipitated with Flag or HA antibodies as indicated and examined for the presence of Raf-1, B-Raf, and 14-3-3 by immunoblotting with the indicated antibodies. As a control for protein expression, lysates were immunoblotted as well. (B) Raf proteins were produced by coupled IVT. As indicated, increasing amounts of purified GST-14-3-3 produced in E. coli were added. Flag-Raf-1 or HA-B-Raf proteins were immunoprecipitated and analyzed by Western blotting with Flag- and HA-specific antibodies. DN 14-3-3, dimerization-negative 14-3-3 mutant.