Figure 4.

Seeding potency of sucrose gradient fractionated brain lysates from homozygous M83 mice. A, Western blot analysis of untransfected and full-length human A53T α-synuclein–transfected HEK 293T cells seeded with sucrose gradient-fractionated homozygous M83 brain lysates. Cells were incubated with sucrose gradient fractions for 72 h and harvested for Western blot analysis. Cell lysates were separated into Triton X-100–soluble and –insoluble fractions by ultracentrifugation at 100,000 × g for 1 h at 4 °C. Twenty μg of protein of the supernatant was loaded onto 4–12% BisTris SDS-PAGE for Triton X-100–soluble fractions and 50 μg of protein from the pellet for Triton X-100–insoluble fractions. A representative Western blotting is shown. B, densitometric analysis of Triton X-100–insoluble pS129 in seeded cells. The results are expressed as the mean ± S.D. (n = 3). C, staining of untransfected (293T) and full-length A53T α-synuclein–transfected (A53T) cells unseeded (−) and seeded with 50% sucrose fraction using antibody pS129 (red) and LCO pFTAA (green). Nuclei were visualized with DAPI (blue). Scale bar, 10 μm.