For a more specific analysis of overexpressed tau between the WT and P301S mice, we used the HT7 antibody that specifically detects human tau. As shown in Fig. 3a and b, the amount of human tau in brain protein homogenates appeared stable up to 7 months post-injection in the AAV-WT group of mice. In contrast, in mice injected with the vector encoding P301S mutant tau, we observed a time-dependent accumulation of human tau whereby P301S tau was significantly increased at 7 months when compared to 1.5 and 3 months (Fig. 3a,b). P301S tau reached nearly a two-fold higher level than WT tau in 7-months old mice. Notably, we noticed the presence of high-molecular weight species of the tau protein in brain extracts of the AAV-P301S mice (Fig. 3a).Figure 3The level of human tau increases over time in the brain of mice injected with AAV-P301S. (a) Detection of overexpressed tau using the human tau-specific HT7 antibody in mouse forebrain homogenates at 1.5, 3 and 7 months post-AAV injection. Actin is used as loading control. *Indicates higher molecular weight human tau species observed in the P301S group of mice. (b) Relative human tau levels measured by western blotting of mouse forebrain homogenates (1.5, 3 and 7 months post- injection). Blots are shown in Supplementary Figure 1. For each animal, human tau values are normalized to actin and plotted as percentage of the mean value for the AAV-WT group at 1.5 months. Statistical analysis: two-way ANOVA followed by Tukey’s post-hoc test, group × time effect F(2,30) = 4.89, **p < 0.01; ***p < 0.001. (c) The graph compares the level of human tau (HT7/TAU-13 AlphaLisa) in total soluble proteins from forebrain homogenates, as well as in the soluble and insoluble fractions following sarkosyl extraction. Samples are derived from AAV-WT and AAV-2P mice, 7 months after AAV injection; n = 8 mice per group.