Typhoon fla7000 gel imager

TEM and PAGE were used to characterize fibrillar αS (S1 Fig). For TEM, 10 μL of aggregated protein samples (from both before and after sonication) were incubated on TEM grids (400-mesh Formvar carbon-coated copper, Electron Microscopy Sciences, Hatfield, PA) for 1 to 2 minutes. Sample solution was wicked with filter paper, and the grid was washed with water to remove any excess material and improve background contrast. Grids were then incubated with 1% (w/v) aqueous uranyl acetate solution (10 μL) for 30 to 60 seconds. Excess uranyl acetate was wicked away with filter paper, and grids were air dried. TEM images were collected using a JOEL JEM 1011 TEM (JEOL, Peabody, MA) (operating voltage 100 kV) equipped with a charge-coupled device camera (ORIUS 832. 10W; Gatan, Pleasanton, CA). The lengths of the PFFs post sonication were quantified using the Fiji measuring tool on TEM images [57 (link)].
For PAGE, aggregated protein solutions were centrifuged to pellet the aggregated material. The supernatant was removed and pellet was resuspended in the starting volume of buffer. Both supernatant and resuspended pellet (20 μL) were loaded on a 4% to 12% polyacrylamide gel. Gels were imaged using a Typhoon FLA7000 gel imager (GE Life Sciences, Pittsburgh, PA) using Coomassie stain mode.

DNA substrate was prepared via PCR and gel extraction of a 233-bp construct containing the Sox2 binding motif engineered into a 601 sequence (Supplementary Table 1) as previously described^{16 (link)}. 10 nM of DNA substrate was incubated with Sox2 and HMGB constructs in T150 buffer at room temperature for 30 min. The reaction mixture was loaded onto a 5% non-denaturing polyacrylamide gel, which was run in 0.5× Tris-Borate-EDTA at 4 °C at 100 V for 90 min, stained with SYBR Gold (Invitrogen), and visualized using a Typhoon FLA7000 gel imager (GE Healthcare).

As an orthogonal approach to the FCS and imaging approached described above, uptake of both monomer and PFF αS were quantified by PAGE. SH-SY5Y cells were incubated with 200 nM αS, as described above for FCS uptake experiments. After incubation for the desired time (1, 3, 5, 8, 12, or 24 hours), the media were removed from cells and stored at 4°C. After all samples were collected, 20 μl of each sample was run on a 4%–12% polyacrylamide gel.
To quantify the amount of protein internalized, an identical set of experiments was carried out, with modifications as described following. For each time point, transferrin-AL488 (100 nM) was added 30 minutes prior to the due time of the time point to serve as a loading control. At the desired time points, the cells were detached from the wells by 0.05% Trypsin-EDTA (Life Technologies Carlsbad, CA), pelleted, and lysed in 250 μl RIPA lysis buffer (Thermo Fisher, Waltham, MA). Cell lysates (20 μl of stock) were run on PAGE gels. Cell lysates (20 μl) were run on 4%–12% polyacrylamide gels.
Gels of both extracellular and internalized αS were imaged using a Typhoon FLA7000 gel imager (GE Life Sciences, Pittsburgh, PA) using fluorescent imaging mode to detect αS-AL488 or transferrin-AL488. Image J was used to quantify the bands [57 (link)].