Figure 4. PINK1 interacts directly with the amino-terminus of Fbxo7.

a, Co-immunoprecipitation of PINK1-Myc and FLAG-Fbxo7 in whole cell lysates from U2OS cells overexpressing both proteins. b, Co-immunoprecipitation of PINK1-Myc with full length and two N-terminal deletions of FLAG-Fbxo7. PINK1-Myc is detected at low levels in complex with FLAG-Fbxo7(89-522) but not with FLAG-Fbxo7(129-522). c, as with (b) using lysates from U2OS cells expressing PINK1-Myc, and FLAG-Fbxo7 containing either N- or C-terminal truncations. d, In vitro pull down experiments were performed using in vitro translated (IVT) PINK1-Myc and either GST or GST fusions of Fbxo7 containing (1-398) or (129-398) immobilised on glutathione beads. e, Competitive binding assays using immobilised GST-Fbxo7(1-398) incubated with IVT FLAG-Parkin and/or full length (top panel) or N-terminally truncated (bottom panel) PINK1-Myc. Input and bead-bound proteins were analysed by immunoblotting as indicated. f, as with (e) immobilised GST-ΔN-PINK1 (top panel), GST-Parkin (middle panel) and immobilised GST alone (bottom panel) were incubated with combinations of IVT FLAG-Parkin, T7-ΔN-PINK1 and Fbxo7-HA as indicated. Input and bead-bound proteins were analysed by immunoblotting with the indicated antibodies. All western blots were performed a minimum of three times. Full-length blots are presented in Supplementary Figure S9.