FIGURE 1.

Introduction of seed α-syn into cultured cells with Lipofectamine reagent. A, purified recombinant α-syn (soluble form; 2 μg) and filaments (2 μg) were sonicated and then incubated with LA. The protein-LA complexes were dispersed in Opti-MEM and added to SH-SY5Y cells. After 14 h of culture, the cells were collected and sonicated in SDS sample buffer. After boiling, the samples were analyzed by immunoblotting with a phosphorylation-dependent anti-α-syn Ser(P)¹²⁹ (PSer129) (right) or a phosphorylation-independent antibody, Syn102 (left). B and C, carboxyl-terminally HA-tagged α-syn fibril seeds (Seed-HA) were transduced into cells by the use of LA. After incubation for 1 day (1d) or 3 days (3d), cells were harvested with or without trypsin, and proteins were differentially extracted from the cells with Tris-HCl (TS), Triton X-100 (TX), and Sarkosyl (Sar), leaving the pellet (ppt). Immunoblot analyses of lysates using anti-HA and anti-Ser(P)¹²⁹ are shown. The immunoreactive band positive for anti-HA or anti-Ser(P)¹²⁹ in the Triton X-100-insoluble fraction was quantified. The results are expressed as means ± S.E. (n = 3). D, confocal laser microscopic analysis of cells treated with Seed-HA in the presence of LA. Cells were transduced with 2 μg of Seed-HA using 5 μl of LA. After a 48-h incubation, cells were fixed and immunostained with anti-Ser(P)¹²⁹ (green) and anti-HA (red) and counterstained with TO-PRO-3 (blue).