Western blot

Cells were plated in 35mm dishes. Where applicable, they were transfected 48h after seeding with the GFP-2A-αSyn-RFP construct. 5 days later they were washed with PBS, trypsinized and pelleted with centrifugation at 1500rpm for 7min. The pellet was frozen until further usage. A pellet from a 35mm dish was lysated in 100ul of lysis buffer consisting of RIPA 1x (#9806S, Cell signaling) and cOmplete protease inhibitors 1x (#11697498001, Merck). For the FACS sorted cells, approximately 200,000 and 1,000,000 cells were lysated in 20ul of lysis buffer for the RFP+GFP- and the RFP+GFP+ populations, respectively. The protein concentration in each sample was measured with the BCA method (#23225, ThermoFisher scientific). A total of 20ug of protein were blotted per sample, after being diluted in 1x NuPage LDS sample buffer (#NP0008, ThermoFisher scientific) and 1x NuPAGE Sample Reducing Agent (#NP0009, ThermoFisher scientific) (final protein concentration 1ug/ul). The samples were run on 4%–12% Bis-Tris gels (#NP0321BOX, ThermoFisher scientific) at 120Volt at room temperature with 1x MES running buffer (#NP0002, ThermoFisher scientific). The gels were transferred onto PVDF membranes using the iBlot 2 dry blotting system. After blocking for 2h at room temperature in 5% SureBlock (#SB232010-50G, Lubioscience) in PBS, the membranes were incubated for 2h at room temperature in mouse anti-αSyn antibody, 1:1000 (#610786, BD Bioscience) in 1% SureBlock. Subsequently, the membranes were washed with PBS + 0.1%Tween and incubated with the secondary HRP conjugated goat anti-mouse antibody 1:12000 (#115-035-003, Jackson laboratories) for 1h at room temperature. After washing, the membranes were treated with Crescendo HRP substrate (#WBLUR0100, Merck) and imaged with the Fusion Solo 7S (witec ag). The membranes were also stained with a mouse anti-actin antibody for 1h at room temperature (#MAB1501R, Merck).