Fig. 3.
Soluble endogenous Syn is recruited into inclusions by fibrils. (A–C) QBI-WT-Syn cells were seeded with fibrils generated by using recombinant α-Syn containing a C-terminal Myc-tag. Double staining for Myc and anti-α-SynpSer129 (pSyn) revealed that fibril seeds form the core of inclusions whereas pSyn predominates in the periphery regions. (D) Immunoblot of detergent-soluble (TriX) and detergent-insoluble (SDS) fractions of cell lysates from control unseeded QBI-WT-Syn cells. (E and F) Lysates from cells transduced with Syn-Myc fibrils contained both WT (black arrowhead) and Syn-Myc (white arrowhead) in the insoluble fraction, indicating that α-Syn originating from the cell comprise the majority of α-Syn within inclusions. A smear representing high molecular weight α-Syn species (**) could also be detected in the SDS fraction. Remaining Syn-Myc seeds within the SDS-fraction of transduced lysates were immunoprecipitated with anti-Myc (9E10). (G) Antibodies against α-Syn (SNL-4) indicate efficient pull-down of Syn-Myc seeds. (H) Probing with anti-pSyn indicates that phosphorylation occurs overwhelmingly in endogenous α-Syn but not exogenous fibrils. (I and J) Inclusions detected in cells stably expressing Myc-tagged α-Syn (QBI-Syn-Myc) transduced with WT α-Syn PFFs. Positive staining for Myc (I) and Syn303 (J) indicates that inclusions contain α-Syn of cellular origin. [Scale bars, 2.5 μm (A); 5 μm (I).] *, IgG light chain; FT, flow-through fraction.