Extracellular α -synuclein is internalized and assembled into SDS-stable oligomers in neuronal and oligodendroglial cells. A, The characterization of recombinant αSYN. (a) The expressed GST-αSYN and tag-free αSYN were analyzed by CBB staining and western blotting with an anti-αSYN Ab (1 μg per lane). Upon IPTG induction, the transformed E. coli produced a GST-αSYN fusion protein that migrated at 44 kDa under denaturing conditions (asterisk). Following the removal of the GST-tag, monomeric αSYN was detected, which corresponded to a molecular mass of 18 kDa (arrowhead). An immunoblot using an anti-GST antibody did not detect any GST-αSYN after the removal of the GST moiety. (b) In the native condition, the absolute majority of recombinant αSYN migrated to approximately 54 kDa, corresponding to trimeric (X3) αSYN as well as some monomers (arrowhead) and oligomers (X2 and X4)/multimers. (c) Recombinant αSYN in cell-free culture medium did not self-assemble into SDS-stable oligomers after 24 hours at 37°C. B, Extracellular αSYN was incorporated and assembled into oligomers in neuronal and oligodendroglial cells. SH-SY5Y (a) and KG1C (b) cells were exposed to 5 μM αSYN for the indicated amount of time, and the cells were then subjected to fractionation and αSYN immunoblot analysis (50 μg lysate per lane). One minute after the addition of αSYN, monomeric αSYN (X1) was incorporated, and the amount of αSYN increased thereafter mainly in the hydrophilic fraction. In parallel, the SDS-resistant dimeric/trimeric αSYN (X2-X3), as well as the multimers and truncated fragments (arrow), gradually appeared in the hydrophilic fractions. Hsp90 and Na+/K+ ATPase α were used as markers for the cytosol and the plasma membrane, respectively. (c) A dose-dependent increase in intracellular monomeric (X1) and oligomeric (X2-X3) αSYN was observed mainly in the hydrophilic fractions prepared from the cells exposed to varying concentrations of recombinant αSYN (0–10 μM) for 24 hours. An asterisk indicates the non-specific band. Representative blots from three independent experiments are presented.