For αSyn quantification, total yeast protein extraction was performed using TBS buffer and glass bead lysis in the presence of protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany)7 ,72 (link). Protein quantification was performed using Micro BCATM Protein Assay Kit (Thermo Scientific, Rockford, USA). After extraction, a western blot was performed following standard procedures. Antibodies used: 1:5000 αSyn (Purified Mouse Anti-α-Synuclein, Clone 42/α-Synuclein (RUO), Catalog Number 610787, BD Transduction Laboratories, San Jose, CA, USA), 1:5000 PGK (Clone 22C5D8, Catalog number 459250, Life Technologies, Paisley, UK), 1:2500 GAPDH (Clone GA1R, Catalog number MA5-15738, Thermo Scientific, Rockford, USA), 1:500 CPY (Clone 10A5B5, Catalog number A-6428, Life Technologies, Paisley, UK).
For Drosophila samples, proteins were extracted in lysis buffer supplemented with Complete Protease Inhibitor Cocktail tablets from Roche (Basel, Switzerland). The protein extracts were obtained from 17 days old flies. Total protein was quantified using the DC Protein Assay, from Bio-Rad (CA, USA). For the western blot, we used anti-GFP (Clone 3H9, Catalog number 3h9-100), from Chromotek and anti-α-tubulin (Clone AA4.3, Catalog number RRID:AB_579793, Developmental Studies Hybridoma Bank, IA, USA).
For Drosophila samples, proteins were extracted in lysis buffer supplemented with Complete Protease Inhibitor Cocktail tablets from Roche (Basel, Switzerland). The protein extracts were obtained from 17 days old flies. Total protein was quantified using the DC Protein Assay, from Bio-Rad (CA, USA). For the western blot, we used anti-GFP (Clone 3H9, Catalog number 3h9-100), from Chromotek and anti-α-tubulin (Clone AA4.3, Catalog number RRID:AB_579793, Developmental Studies Hybridoma Bank, IA, USA).
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