FIG. 5.

MAP kinase activation. (A) MAP kinase assay. Proteins (400 μg) from lysates of transfected cells deprived of IL-3 for 12 h were immunoprecipitated with anti-Erk1 and anti-Erk2 antibodies and assayed for kinase activity, using myelin basic protein (MBP) as the substrate. As a positive control, 12-h IL-3-starved cells were stimulated for 10 min with IL-3 and assayed for kinase activity. The amounts of immunoprecipitated Erk1 and Erk2, shown below, were determined by subjecting the same filter to Western blotting (WB) with anti-Erk antibodies. (B) Phosphorylation of MAP kinase. Protein (1,300 μg) from lysates prepared as described for panel A were immunoprecipitated with anti-Erk1 and anti-Erk2 antibodies. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting with antibodies specifically recognizing activated phosphorylated Erk.