Fig. 1: Characterization of the V40G variant of α-synuclein.

a CD spectroscopy of human WT-syn monomer, V40G-syn monomer, aged WT-syn, and V40G. b Comparison of CD data at 218 nm. c TEM image of human aged WT-syn (top) and V40G (bottom). Scale bars, 0.4 μm. d Thio T binding kinetics of WT-syn or V40G recombinant α-synuclein over 9 days. e Dye binding assays of fresh/aged WT-syn and V40G. f Size exclusion chromatography (top) and western blotting (bottom) of aged V40G. Cyto C: Cytochrome C. g Size exclusion chromatography (top) and western blotting (bottom) of the V40G monomer. Cyto C: Cytochrome C. h Ultracentrifugation assay. Western blotting (top) and quantification (bottom). i Western blots and quantification of WT-syn and V40G without (left) or with PK digestion (right). Note that the size ranges of the left and right blots are different. j Western blotting of PMCA end-products without PK digestion. Human-aged WT-syn or V40G was used as a seed, and human fresh WT-syn was used as a substrate for the PMCA reaction. k Western blotting of PMCA end-products with PK digestion. l, m Thio T binding kinetics of recombinant human α-synuclein with human aged WT-syn or aged V40G as seeds. In all panels, “fresh” indicates pure monomers, and “aged” indicates the protein samples incubated for 9 days at 37 °C with constant agitation. m The results of the Thio T binding assay were confirmed by western blotting. n, o X-34 binding kinetics of recombinant mouse α-synuclein with aged human WT-syn or aged human V40G as seeds. In all panels, “fresh” indicates pure monomers, and “aged” indicates the protein samples incubated for 3–4 days at 37 °C with constant agitation. o The result from the X-34 binding assay was confirmed by western blotting. Significance was assessed by one-way ANOVA with Tukey’s post hoc comparison between groups (b) or by two-tailed unpaired Student’s t test (e, i), *P < 0.05, **P < 0.01, ***P < 0.0001. All data are presented as the mean ± SEM.