Figure 2.

Domain analysis of the parkin region that interacts with 14-3-3η. (A) Schematic representation of WT parkin and its deletion- and disease-related missense mutants. See text for the domain structures of parkin and mutants. The dotted line denotes the deleted region. (B) Interaction between 14-3-3η and parkin mutants. FL-parkin (2 μg) or its mutant (10 μg) plasmids were transfected into HEK293 cells, as described in Figure 1D. The cell lysates (200–600 μl) were immunoprecipitated with anti-FL-antibody beads. Note that various amounts of the lysates were used to adjust roughly the levels of expressed parkin mutants. The resulting immunoprecipitates were mixed with other cell lysates (200 μl) prepared from cells that had been transfected with Myc-14-3-3η plasmid (2 μg) and incubated for 6 h at 4°C. Then, the extensively washed immunoprecipitates and cell lysate (7.5% input) were analyzed by Western blotting with anti-Myc and FL antibodies. Asterisks denote nonspecific bands.