Fig. 3

Seeding of Tau aggregation with sucrose gradient fractions from the brains of mice transgenic for human mutant P301S Tau in a cell-based assay. The mice were aged 4.4 weeks (no symptoms, no Tau filaments) or 24.4 weeks (symptoms, abundant Tau filaments). Sucrose gradient fractions were used to seed aggregation of Tau in HEK cells expressing 1N4R Tau with the P301S mutation. The pellet from a 100,000 g spin of seeded cells was analysed by Western blotting for total Tau and Tau phosphorylated at S202/T205 (anti-Tau antibodies DA9 and AT8). Filamentous Tau runs at approximately 68 kDa (HMW, high-molecular weight); non-filamentous Tau runs at approximately 59 kDa (LMW, low-molecular weight). The positive control consisted in seeding with sarkosyl-extracted Tau from unfractionated brains of symptomatic transgenic P301S Tau mice and the normalised positive control was seeding with sarkosyl-extracted Tau from symptomatic mice, normalised for total Tau levels relative to those of the sucrose gradient fractions. Seeding ability correlated with the presence of the 64 kDa band in 24.4-week-old mice (20–50% sucrose gradient fractions). No seeding was observed upon addition of sucrose gradient fractions from the brains of 4.4-week-old mice