Fig. 4. Effect of β-arrestin-1 mutants on GRK2-K220R degradation. (A) HEK-293 cells were transiently transfected with GRK2-K220R, β₂AR and wild-type β-arrestin-1 or the indicated β-arrestin-1 mutants, and the turnover of GRK2-K220R was analyzed as in Figure 2. Data are the mean ± SE of at least four independent experiments performed in duplicate. Representative gel fluorographs are shown below. Cell lysates were also subjected to immunoblot analysis with anti-β-arrestin antibodies as detailed in Materials and methods to assess the expression of the different β-arrestin constructs (lower panel). (B) Similar pulse–chase experiments were performed in cells transfected with wild-type GRK2, β₂AR and the indicated β-arrestin-1 constructs, incubated for 1 h in the presence (+) or absence (–) of 10 µM isoproterenol (ISO). The gel fluorographs are representative of three independent experiments. (C) Cos-7 cells were transiently transfected with GRK2, β₂AR and β-arrestin-1 mutants or empty vector as indicated, and GRK2 turnover under basal conditions analyzed as in previous figures. Data are the mean ± SE of three independent experiments. Representative gel fluorographs are shown below.