FIG. 7.

ERK2 phosphorylates c-Fos in vitro and in vivo. (A and B) Bacterially expressed c-Fos TADs, six-His c-FosTAD-wt, and six-His c-FosTAD-m were purified and assayed as substrates for an immunoprecipitated (A) or recombinant purified (b) preparation of ERK2. In vitro kinase assays were performed as described in Materials and Methods. The purified six-His substrates (A and B, lower panels) and the HA-tagged ERK2 (A, middle panel) used in the kinase reactions were detected by Western blotting with the indicated antibodies. (C) In vitro cold kinase assay using purified six-His c-Fos TAD as a substrate for recombinant ERK2. Reaction products were analyzed by Western blotting using anti-c-Fos antibodies or anti-phospho-T/P antibodies. (D) HEK-293T cells were transfected with pCEFLc-Fos (1 μg/well) alone or in combination with ERK2+MEKEE (0.5 μg each). Total lysates were analyzed by Western blotting using anti pCEFL-AU5-c-Fos antibodies (left panel) or immunoprecipitated with anti-c-Fos and immunoblotted using anti-phospho-T/P antibodies (right panel).