Ubiquitin Proteomics in Drosophila

UAS-BirA and UAS-(^BioUb)₆-BirA [39 (link)] and their recombination with GMR-GAL4 flies for the study of ubiquitin proteomics has been previously described [43 (link)]. FLAG-tagged Parkin wild-type (Parkin^WT) and Parkin^LD flies were generated at Bestgene using the pUASattb constructs described above. Both UAS-Parkin^WTand UAS-Parkin^LD lines were independently crossed with GMR-GAL4,UAS-(^BioUb)₆-BirA to finally generate: GMR-GAL4,UAS-(^BioUb)₆-BirA/CyO;UAS-Parkin^WT and GMR-GAL4,UAS-(^BioUb)₆-BirA/CyO;UAS-Parkin^LD. GMR-GAL4/CyO;UAS-BirA/TM6 and GMR-GAL4,UAS-(^BioUb)₆-BirA/CyO flies were additionally used as controls. UAS-GFP, elav-GAL4, GMR-GAL4, Tub-GAL4, Da-GAL4, Ple-GAL4 flies were obtained from Bloomington Drosophila Stock Center. UAS-GFP^CL1 flies were obtained from [48 (link)] and park²⁵/TM6b GFP-w⁺ and UAS-park were obtained from [16 (link)]. Flies were grown in 12 h light-dark cycles at 25 °C and were fed with wheat flour and yeast food (1% agar, 5.5% dextrose, 3.5% wheat flour, 5% yeast, 0.25% Nipagen, 0.4% Propionic acid and 0.02% Benzalkonium Chloride in distilled H₂O).