Figure 6.

PRKN/PARK2 is identified as a PTENα-associated protein upon mitochondrial depolarization. (a) Silver staining and MS analysis of PTENα-associated proteins. The empty vector (PSA), S•Tag PTENα and -PTEN were purified from HEK293T cells and incubated with mouse cardiac homogenate; associated proteins were identified by mass spectrometry. (b) Co-IP with HEK293T cells co-transfected with plasmids encoding PTENα-GFP and PRKN-FLAG. Cells were treated with 10 μM CCCP or DMSO for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. The asterisk indicates degradation of PTENα. (c) Co-IP in HEK293T cells co-transfected with plasmids encoding PRKN-GFP and FLAG-PTENα. Cells were treated with 10 μM CCCP or DMSO for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. (d) In vitro binding assay of PTENα-His with GST-PRKN. PTENα-His (1 µg) and 3 μg GST-PRKN were mixed in PBS containing 0.1% NP40, and PRKN was immunoprecipitated with a PRKN monoclonal antibody or mouse lgG and immunoblotted with anti-PTEN or anti-PRKN. PTENα-His was expressed and purified from insect cells, and GST-PRKN was expressed and purified from E. coli. The asterisk indicates degradation of PTENα. (e) Interaction of FLAG-PTENα and its truncations with PRKN-GFP in HEK293T cells with CCCP treatment. Cells were treated with 10 μM CCCP for 1 h before harvest. Lysates immunoprecipitated with anti-FLAG were immunoblotted with anti-GFP or anti-FLAG. IgH, heavy chain of IgG. (F) Interaction of PRKN-GFP and its truncations with HA-PTENα in HEK293T cells with CCCP treatment. Cells were treated with 10 μM CCCP for 1 h before harvest. Lysates immunoprecipitated with anti-GFP were immunoblotted with anti-HA or anti-GFP.