Fig. 2
From: Quantitative proteomic analysis of Parkin substrates in Drosophila neurons
Proteomic analysis of Parkin substrates in Drosophila melanogaster neurons. a Scheme of the strategy used to identify proteins ubiquitinated by Parkin in Drosophila neurons. FLAG-tagged Parkin WT or LD flies also expressed biotin modified ubiquitin (BioUb) in Drosophila photoreceptors using GMR-GAL4 driver (image shows GFP expression with GMR-GAL4 in an adult fly head). BioUbiquitinated material was purified using Neutravidin beads and isolated material was analysed by Mass Spectrometry (MS). Ubiquitinated proteins enriched in Parkin WT over-expressing neurons were then identified based both on protein LFQ levels and peptide intensities. b Anti-Parkin immunoblotting shows Parkin levels in head extracts of GMR-GAL4/CyO;UAS-BirA/TM6 (C), GMR-GAL4,UAS-( Bio Ub) 6 -BirA/CyO (Ub), GMR-GAL4,UAS-( Bio Ub) 6 -BirA/CyO;UAS-Parkin WT (WT) and GMR-GAL4,UAS-( Bio Ub) 6 -BirA/CyO;UAS-Parkin LD (LD) flies. Parkin null (park 25) flies were used as a negative control. WT and LD samples were diluted 100 times. c Anti-BirA western blot of C, Ub, WT and LD heads extracts. d Anti-Biotin western blot of C, Ub, WT and LD heads extracts. e C, Ub, WT, LD fly heads were subjected to BioUb pulldown. Input (1/100), Flowthrough (FT, 1/100) and Elution (1/400) were immunoblotted for biotin. f Silver staining of C, Ub, WT, LD BioUb pulldown elutions (undiluted). In panels B through F, arrowheads indicate ectopic Parkin; arrow indicates endogenous Parkin; smear highlighted by the brace represents BioUbiquitinated material; and the asterisk shows an endogenously biotinylated protein