Fig. 3.

In vitro characterization of patient-derived αSYN assemblies. a Electron micrographs of αSYN assemblies obtained after the 3rd cycle of amplification by PMCA from PD, MSA and DLB patients and from de novo generated αSYN Fibrils and Ribbons. Scale bar = 200 nm. b Limited proteolytic patterns of αSYN assemblies obtained after the 3rd cycle of amplification by PMCA from PD, MSA and DLB patients and from de novo generated αSYN Fibrils and Ribbons. Monomeric αSYN concentration is 100 µM. Proteinase K concentration is 3.8 µg/ml. Samples were withdrawn from the reaction before PK addition (lane most to the left), immediately after PK addition (second lane from left) and at time 1, 5, 15, 30 and 60 min from left to right in all panels. PAGE analysis was performed as described in the “Materials and methods” section and the gels were stained with Coomassie blue. The molecular weight markers are shown on the left. c The conformational FILA4 antibody distinguishes equal amounts (0.2 µg) of PMCA-amplified αSYN strains spotted on a single nitrocellulose membrane. d Quantification of FILA4 signal for PD, MSA, DLB, and reference samples (Fibrils, Ribbons, and monomeric αSYN). Data represent mean ± SE (*p < 0.05 for one-way ANOVA, n = 4–6 PD- or MSA-amplified strains versus DLB-amplified strain)