Fig. 2

Establishment and characterization of the aggregated α-Syn ECL assay system. a The aggregated α-Syn standard curve was generated over a range of 9 pg/ml to 10 ng/ml (black line; R² = 0.999). Specificity was tested by measuring unphosphorylated (red line) or phosphorylated (blue line) monomeric species run in the same aggregated α-Syn assay. b Linearity-of-dilution of the aggregated α-Syn assay was assessed by using dilutions of 1:1000, 1:100 and 1:10 in the cytosolic and membrane fractions. c Spike–in recovery of the aggregated α-Syn assay was tested by spiking 250, 500, and 1000 pg/ml of α-Syn aggregates into the cytosolic fraction or 50, 100, and 200 pg/ml into the membrane fraction. d Specificity of MJFR14 conformational specific antibody was examined. Red line: aggregated α-Syn signals after dissociation using 8 M Urea treatment. Green line: aggregated α-Syn standard curves incubated with 1 mM Urea, the same final concentration as included in the disaggregated calibrator assay. Yellow line: Aβ oligomers detected using the aggregated α-Syn assay (MJFR14 antibody and anti-α-Syn detection antibody). Blue line: Aβ oligomers captured with the conformation-specific α-Syn antibody and an Aβ-specific detection antibody. e The total α-Syn concentrations (measured by using the total α-Syn assay) before and after immunoprecipitation using MJFR 1(recognizing “total” α-Syn, including monomeric and oligomeric/aggregated forms) or MJFR 14 (recognizing aggregated α-Syn only) in erythrocyte samples. f The aggregated α-Syn concentrations (measured by using the aggregated α-Syn assay) before and after immunoprecipitation using MJFR 1or MJFR 14 in erythrocyte samples