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1 doi: /nature10324 Fig. S1: Two dimensional IEF/SDS-PAGE/Western blot analysis of RBC lysate crosslinked with 4 mm BS 3. The blot was probed with αsyn antibody C20. Fig. S2: SDS-PAGE/silver stain analysis of the three stages of αsyn purification from human RBC lysates via (NH 4 ) 2 SO 4 precipitation and hydrophobic interaction chromatography (HIC). The silver band at ~14.8 kda (lane 3) immunoreacted positively for αsyn and negatively for hemoglobin α-chain (not shown). 1

2 Fig. S3: Mean residue ellipticity of purified αsyn tetramers from human RBC with vs. without Lipidex 1000 treatment (overnight, 37ºC). CD spectra were taken at 2.5 µm tetramer protein concentration and 20ºC in 10 mm PO 4 buffer. 2

3 Fig. S4: MALDI-TOF mass spectrometry traces of intact recombinant αsyn (A) and αsyn purified from human RBC (B). Expected mass for recombinant αsyn: 14,460 kda; expected mass for N-acetylated αsyn: 14,502 kda 3

4 Fig S5: Clear Native-PAGE analysis of the migration behavior of human αsyn dependent on conformation. A: Western blot (antibody C20) of M17D cell lysate and recombinant αsyn. B: Coomassie stain of folded RBC αsyn, unfolded (denatured) RBC αsyn, and recombinant unfolded αsyn (purification of RBC samples utilized Sepharose 6B chromatography ; folding states were confirmed by CD spectroscopy). 4

5 Fig. S6: Clear Native-PAGE/Western blot analysis of RBC and 3D5 lysates displaying the tetrameric assembly state of human αsyn in both cell types, consistent with their approximate comigration with purified human transthyretin (tetramer MW = 55 kda ), which is known to occur as a tetramer. Fig. S7. Left: Representative large-angle dark-field cryo-stem image of purified αsyn from overexpressing 3D5 cells. A few representative particles are circled. As a size standard, tobacco mosaic virus (TMV) helical rod was included during EM specimen 5

6 preparation. Right: Mass histogram (bin size 5 kda) of 3,000 automatically selected αsyn particles. Fig. S8: Mean residue ellipticity of αsyn from the stable human neuroblastoma cell line 3D5 (purified by (NH 4 ) 2 SO 4 precipitation, anion exchange and size exclusion chromatography) and from human RBC (purified by (NH 4 ) 2 SO 4 precipitation, hydrophobic interaction and size exclusion chromatography). Both spectra exhibit significant amounts of α-helical structure. Spectra were taken at 15 µm for the RBC tetramer and 3 µm for the 3D5 cell tetramer at 20ºC in 10 mm PO 4 buffer. 6

7 Fig S9: Comparative CD spectra (mean residue ellipticity) of intrinsic αsyn from human neuroblastoma cell line 3D5 and bacterial recombinant monomeric αsyn added extrinsically ( spiked ) in comparable ratios onto the untransfected parental cell line M17D. For both samples, the cells were lysed by sonication, and αsyn was purified identically via anion exchange (AX) and size exclusion chromatography (SEC). Spectra (in 10 mm PO 4 buffer at 20ºC) were taken at 10 µm for the spiked recombinant αsyn monomer and 3 µm for the 3D5 cell αsyn tetramer. Fig S10: A: SPR sensorgram of a dilution series of the native αsyn tetramer isolated from human RBC. The protein was injected on a L1 chip covered with a PC/PS 4:1 membrane. B: Steady-state responses of RBC αsyn tetramer injections plotted against αsyn tetramer concentrations and binding model fit. 7

8 Fig S11: Melting curve of purified tetrameric αsyn (6 µm in 10 mm PO 4 ), monitored via CD spectroscopy, showing the irreversible heat denaturation of the helical folding 8