Extended Data Fig. 1: Biochemical analysis of GCI-α-Syn and LB-α-Syn.

a, Schematic for sequential extraction of brains with α-synucleinopathy. Diseased brain samples were sequentially extracted with buffer of increasing extraction strengths (1% Triton X-100 followed by 1% sarkosyl) to remove soluble proteins. b, Proteinase K-digested GCI-α-Syn and LB-α-Syn were immunoblotted with a series of antibodies targeting specific domains of α-Syn that spanning the entire molecule. c, Summary of the results for experiments described in b. d, Thermolysin-digested and undigested sarkosyl-insoluble fractions from three cases of Lewy body disease (LB1–LB3) and three cases of MSA (GCI1–GCI3) were resolved on 12% Bis-Tris gel and immunoblotted with an antibody against α-Syn (Syn211). e, Sarkosyl-insoluble fractions from a pair of cases (one of Lewy body disease and one of MSA) were incubated with increasing concentrations of thermolysin (with a ratio of thermolysin to total protein that ranged from 1.25 × 10⁻² to 5 × 10⁻²) and immunoblotted with antibody against α-Syn (Syn211). Undigested fractions were loaded on the same gel. f, Trypsin-digested and undigested sarkosyl-insoluble fractions from three cases of Lewy body disease (LB1–LB3) and three cases of MSA (GCI1–GCI3) were resolved on 12% Bis-Tris gel and immunoblotted with an antibody against α-Syn (Syn211). g, Sarkosyl-insoluble fractions from a pair of cases (one of Lewy body disease and one of MSA) were incubated with increasing concentrations of trypsin (with the ratio of trypsin versus total protein range from 1.25 × 10⁻² to 5 × 10⁻²) and immunoblotted with an antibody against α-Syn (Syn211). Undigested fractions were loaded on the same gel. The experiments shown in b and d–g have been repeated three times with similar results. For gel source data, see Supplementary Fig. 1.