FIGURE 7.

Detection of a ∼122-kDa complex consisting of GRK2 and RKIP in mouse hearts. A, mouse hearts were perfused with the cross-linking agent PFA (4% (w/v) in PBS, 7 min) and lysed. Protein complexes were separated by SDS-PAGE and subjected to Western blot with the indicated antibodies. The positions of molecular mass standards are marked on the left. n = 5 independent experiments. B, immunoblot analysis of RKIP phosphorylation in murine heart lysates treated without or with protein phosphatase 1 (PP1; 20 units of PP1/100 μg of lysate; 30 min, 30 °C) for dephosphorylation using antibodies directed against pRKIP(S153). n = 4 independent experiments. C, perfusion of mouse hearts with the PKC inhibitor GFX (1.5 μm; 3 min) and lysed. RKIP phosphorylation was analyzed by Western blot analysis. n = 4 independent experiments. D, mouse hearts were perfused with GFX (1.5 μm; 3 min) followed by perfusion with PFA (4% (w/v) in PBS, 7 min). Protein complexes were separated by SDS-PAGE and subjected to Western blot with the indicated antibodies. The positions of molecular mass standards are marked on the left. Open arrow indicates a nonspecific anti-Raf1 immunoreactive band. n = 4 independent experiments.