FIG. 1.

(A) Raf_Δ26–303-Cx phosphorylates MEK in an in vitro kinase assay. A3.01 T cells were transiently transfected with expression plasmids containing Raf_Δ26–303, Raf_Δ26–303-Cx, kinase-dead mutants (Raf_Δ26–303-KD, Raf_Δ26–303-KD-Cx) or with the empty expression vector as a control (mock). At 42 h posttransfection, cells were stimulated with TPA (25 ng/ml) for 20 min or left untreated. The cells were lysed in RIPA buffer, epitope-tagged Raf kinases were immunopurified with anti-HA (clone 12CA5) antibodies, and an in vitro kinase assay was performed with recombinant MEK as the substrate (for details, see Materials and Methods). Proteins were separated by SDS-PAGE and visualized by autoradiography. (B) Corresponding immunoblots demonstrating that equal amounts of the Raf kinase mutants are expressed in A3.01 T cells.