Fig. 1.

Parkin interacts with c-Abl. (A) GST-parkin immobilized on glutathione-Sepharose beads pulls down recombinant c-Abl. Pull downs were immunoblotted with anti-c-Abl. (B) Mouse brain lysates were immunoprecipitated with anti-parkin and anti-IgG, followed by immunoblot with anti-parkin or anti-c-Abl antibodies. (C) Lysates from SH-SY5Y cells transfected with GFP-c-Abl or a kinase-dead (KD) (lysine 290 arginine) version of c-Abl (GFP-c-Abl-KD) and V5-parkin were subjected to immunoprecipitation (IP) with anti-GFP, followed by anti-V5 immunoblotting (Middle) or with anti-GFP antibody (Bottom) to show an equivalent amount of immunoprecipitated c-Abl. (D) Lysates from SH-SY5Y cells transfected with V5-parkin and GFP-c-Abl and GFP-c-Abl-KD subjected to IP with anti-V5, followed by anti-GFP (Middle) or anti-V5 (Bottom) immunoblotting to show an equivalent amount of immunoprecipitated parkin. (E) Lysates from SH-SY5Y cells transfected with V5-parkin and c-Abl domain constructs were subjected to IP with anti-V5, followed by anti-c-Abl (Middle) or anti-V5 (Bottom) immunoblotting. The arrow highlights the F3 domain of c-Abl. The deletion domains of c-Abl used are shown at the bottom of the panel. (F) Lysates from SH-SY5Y cells transfected with GFP-c-Abl and myc-tagged fragments of parkin subjected to IP with anti-myc antibodies, followed by anti-GFP (Middle) or anti-myc (Bottom) immunoblotting. A schematic representation of the different parkin fragments used is shown. All experiments were repeated at least three times and representative images of the immunoblots are shown.