Additional file 7: Figure S7. of Quantitative proteomic analysis of Parkin substrates in Drosophila neurons

Over-expression of hParkin (WT) induced an increase of VPS35 ubiquitination compared to control (C) or to over-expression of inactive hParkin (LD) in four independent experiments. (A) The complete gels of Fig. 8a showing four independent in vivo ubiquitination assays for VPS35 in SH-SY5Y cells. Ubiquitination of YFP-tagged VPS35 was analysed by Western blot after capture of the YFP-tagged protein. Mouse anti-GFP antibody was used for detecting the captured VPS35 (shown in green), and HRP-conjugated anti-FLAG antibody for monitoring its ubiquitinated fraction (shown in red). Untagged Parkin over-expression levels in the whole cell extracts are monitored with anti-Parkin antibody. (B) Quantification of the ubiquitination status of VPS35 relative to the non-modified form was performed calculating the ratio FLAG:GFP with Image-J. The plot shows relative levels of VPS35 ubiquitination normalized to the GFP levels. (PDF 14700 kb