FIGURE 3.

RKIP lacking amino acids 143–146 has a high propensity for dimerization and GRK2 binding. A and B, immunoprecipitation (IP) of wild-type FLAG-RKIP (wt) or FLAG-RKIP^Δ143–146 (RKIP^Δ143–6) from lysates of HEK293 cells treated with TPA (1 μm, 5 min) as indicated. Co-precipitated wild-type Myc-RKIP (A), Myc-RKIP^Δ143–146 (A), GRK2 (A), and Raf1 (B) were detected by Western blot analysis with the respective antibodies. The lower molecular mass of the deletion mutant RKIP^Δ143–146 is visible due to the usage of 17% (w/v) acrylamide SDS-PAGE gels. C, immunoblot analysis (IB) of the respective cell lysates of A with anti-pRKIP (Ser-153, S153) antibodies. D, immunoblot analysis of Ser-153 phosphorylation of overexpressed RKIP^Δ143–146 in HEK293 cells pretreated with either TPA (1 μm, 5 min) or bisindolylmaleimide I (GFX; 3 μm, 2 h) as indicated. E, immunoprecipitation of FLAG-RKIP^Δ143–146 from cells pretreated with either TPA (1 μm, 5 min) or GFX (3 μm, 2 h) as indicated with subsequent immunoblot analysis of co-immunoprecipitated Myc-RKIP^Δ143–146 or GRK2. n = 4–7 independent experiments.