α synuclein clone 42

Human V5-tagged VPS35 plasmids were obtained from Addgene (no. 21691; ref. 58 (link)) and previously described (18 (link)). Mouse VPS35 cDNA was obtained from Dharmacon. Primary antibodies used were VPS35 (ab57632 or ab154647; Abcam), VPS26 (ab23892; Abcam), VPS29 (ab10160; Abcam), WASH1 (Sigma), Fam21A-D (S-13; Santa Cruz), V5 and V5-HRP (Life Technologies), TH (NB300-109; Novus Biologicals or clone TH-2; Sigma), α-synuclein (clone 42; BD Biosciences), pSer129-α-synuclein (clone EP1536Y; Abcam), APP (clone 22C11; Millipore), tau (clone Tau5; Millipore), phospho-tau (clones AT8, PHF1, CP13, and MC1, provided by Peter Davies, Feinstein Institute for Medical Research, New York), NeuN (ABN78; Millipore), Iba1 (Wako), GFAP (DAKO), and β-tubulin (clone TUB 2.1; Sigma). Secondary HRP-conjugated IgG, light chain-specific (Jackson Immunoresearch), biotinylated IgG (Vector Labs), and AlexaFluor-488 or -594 IgG (Thermo Fisher) were used.

Fly heads were dissected then homogenized in 2× Laemmli buffer, boiled for 10 min, and centrifuged. SDS-PAGE was performed (Lonza) followed by transfer to nitrocellulose membrane (Bio-Rad) and microwave antigen retrieval in PBS. Membranes were blocked in 2% milk in PBS with 0.05% Tween-20 for 1 h, then immunoblotted with appropriate primary antibody in 2% milk in PBS with 0.05% Tween-20 overnight at 4 °C. Primary antibodies used include α-synuclein H3C (1:10,000 to 1:100,000, mouse, Developmental Studies Hybridoma Bank), α-synuclein clone 42 (1:5000 mouse, BD Bioscience), and phospho-serine 129 α-synuclein (1:5000, rabbit, Abcam). Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:50,000) in 2% milk in PBS with 0.05% Tween-20 for 3 h. Signal was developed with enhanced chemiluminescence (Thermo Scientific).