Journal: Molecular Neurodegeneration

Article Title: Quantitative proteomic analysis of Parkin substrates in Drosophila neurons

doi: 10.1186/s13024-017-0170-3

Figure Lengend Snippet: Human Parkin and VPS35 interaction: VPS35 is ubiquitinated by wild type Parkin in human cells. a VPS35 is ubiquitinated by untagged WT Parkin in SH-SY5Y cells. YFP-VPS35 showed a significant increase in its ubiquitinated fraction when it is over-expressed with wild type untagged hParkin (WT), as shown with anti-FLAG (to FLAG-Ub) antibody Western blot ( red ) compared to control (C) or the single point (C431S) mutated hParkin (LD). The non-modified form of VPS35 was detected by GFP antibody ( green ). The bottom panel shows levels of Parkin protein in the whole cell extract before the isolation of the GFP-tagged proteins. The endogenous Parkin protein is barely detected. 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. Statistical significance in Western blotting semi-quantification was evaluated using an analysis of variance (ANOVA) complemented by Tukey’s honest significance difference test (Tukey’s HSD) performed in GraphPad PRISM software. Statistical significance differences (**, p < 0.01 (mean ± SEM, n = 4)) were observed for the WT Parkin sample relative to both control and LD Parkin samples. For LD Parkin sample ns indicates not significant differences relative to the control sample. c VPS35 knockdown does not affect Parkin - dependent mitophagy. hTERT-RPE1 cells stably over-expressing YFP-Parkin were subjected to control and VPS35 siRNA for 72 h or 165 h and treated with 10 μM CCCP for 2, 8 and 24 h. Samples were immunoblotted as indicated. High exp: high exposure

Article Snippet: Statistical significance in Western blotting quantification was evaluated using an analysis of variance (ANOVA) complemented by Tukey’s honest significance difference test (Tukey’s HSD) performed in GraphPad PRISM software.

Techniques: Western Blot, Control, Modification, Isolation, Ubiquitin Proteomics, Software, Knockdown, Stable Transfection, Expressing