PMCA reactions seeded with α-Syn or α-SynFvaggregates. (A) Illustration of PMCA method used to amplify α-Syn or α-SynFv aggregates in the presence of homologous soluble proteins (see Materials and Methods). (B) α-Syn aggregates or α-SynFv aggregates induced by AP20187 were diluted 20 times into freshly prepared soluble proteins of the same species. Supernatant (S, soluble) and pellet (P, insoluble) fractions recovered from centrifugation of 3 μg protein samples withdrawn at the end of each PMCA cycle were analyzed by SDS-PAGE and Coomassie blue staining. This result is representative of three independent experiments. (C) α-Syn or α-SynFv aggregates were diluted 1/20 into soluble homologous proteins and immediately centrifuged. The absence of detectable protein in the pellet fraction indicates the lack of amplification of aggregates. (D) α-Syn or α-SynFv aggregates were submitted to two PMCA cycles with incubations performed under stationary conditions.