α-Synuclein PMCA

α-Synuclein PMCA was performed as described previously (Herva et al., 2014). PCR tubes containing α-synuclein were briefly spun in a benchtop centrifuge (Microfuge^®16 centrifuge, Beckman Coulter), 37±3 mg beads (1.0 mm zirconia/silica beads, BioSpec Products) were added and tubes were sealed with Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA). Samples were placed into a 96-tube rack in a microplate cup-horn sonicator (Misonix 4000) that was filled with water using a circulating water bath to maintain a steady-state temperature of 37°C. PMCA reactions using α-synuclein consisted of 20 s of sonication every 29 min 40 s for varying lengths of time. Power setting 7 was used for all α-synuclein PMCA experiments. For protease resistance studies, PK was diluted in PBSN to 6× the desired final concentration and 4 μl was added to 20 μl of the PMCA product prior to incubation at 37°C for 30 min with occasional agitation. Replacement of PK with PBSN in equivalent samples acted as non-PK controls and these samples were kept at room temperature (RT) during the digestion of PK-containing samples. All samples were then diluted in 4× NuPAGE LDS sample buffer (Life Technologies) and boiled at 100°C for 10 min. Samples were resolved using SDS-PAGE and western immunoblotting was employed to detect α-synuclein species using the antibody MJFR1 (ab138501, Abcam, Cambridge, UK; 1:10,000 dilution) (Brudek et al., 2016). To assess insoluble load of protein exposed to PMCA, ultracentrifugation was used. Following a PMCA, the reaction sample was diluted in PBSN to fill a 200-μl-capacity thick-walled polyallomer tube (Beckman-Coulter). Samples were spun at 436,000 g for 1 h at RT. The pellet containing insoluble protein was washed in PBSN and centrifuged again under the same conditions. The supernatant was then discarded and the pellet resuspended in 8 M urea/PBSN and incubated for 30 min at RT. Western immunoblot analysis to detect human α-synuclein was performed as described previously.