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. Author manuscript; available in PMC: 2014 Oct 16.
Published in final edited form as: Neuron. 2013 Sep 26;80(2):402–414. doi: 10.1016/j.neuron.2013.07.046

Figure 7. Anti-tau antibody treated P301S mice have decreased tau seeding activity in cortical extracts as detected by FRET assay.

Figure 7

(A) Tau seeding activity was measured with RAB soluble fractions of all PBS (N=16), HJ3.4 (N=8), HJ8.5 (N=13), HJ9.3 (N=15), and HJ9.4 (N=13) treated mice on HEK293 cells by FRET assay. HEK293 cells were co-transfected with RD (ΔK280)-CFP and RD (ΔK280)-YFP. 18 hrs later, RAB soluble fractions were added to cells. Seeding activity was significantly reduced in HJ8.5, and HJ9.3 antibody treated mice compared to the PBS or HJ3.4 antibody treated mice. RAB soluble fractions from HJ9.4 antibody treated mice did not have decreased seeding activity compared to the PBS or HJ3.4 antibody RAB soluble fractions. ***p<0.001, Values represent mean ± SEM. (B) RAB soluble fractions from tau KO, PBS-treated, and the anti-tau antibody treated mice were incubated with un-conjugated protein-G-agarose beads at 4°C with end-over-end rotation for 24 hours. This precipitates any residual antibody in the brain, including antibody bound to tau seeds. Elution of any seeding activity from the antibody/bead complexes was measured by FRET assay. There was significantly less seeding activity observed in HJ8.5 and HJ9.3 antibody treated mice versus PBS-treated mice ****p<0.0001, values represent mean ± SEM. (C) 70% FA fractions of 9 month old P301S brain cortex region of all treated groups analyzed by ELISA showed a strong correlation with FRET analysis performed with the RAB soluble fractions. (D) Comparison between tau levels (X-axis) and seeding activity (Y-axis) present in RAB soluble fractions of 9 month old P301S brain cortex of all treated mice assessed. There was no significant correlation between these 2 measures. (E) Tau species in the RAB soluble fractions of 3 month old knockout (KO), 3 month old wild type (WT), 3 month old P301S, and 9 month old PBS-treated P301S mice were separated on SDD-AGE, followed by western blotting. Polyclonal mouse anti-tau antibody was used for detecting tau species. High molecular weight tau species present in the RAB soluble fraction in both 3 month old P301S mice and larger amounts present in 9 month old P301S mice.