Fig. 3: Overexpression of USP10 induces Tau aggregation in primary neurons.
a A Schematics of the experimental design. b Primary neurons were infected with AAV9-FLAG-USP10 on DIV4, MOI = 105, and 7 days later, samples were extracted and subjected to western blotting. c–e Graph showing normalized protein levels of Flag-USP10, pS199, AT8, and Tau5. f Immunocytochemistry showed the distribution of AT8 expressing USP10 compared to control. Scale bars, 50μm, inset also 50 μm. g Quantification of the mean of integrated optical density (Mean IOD), 3-10 cells per field of view (FOV) were counted, and five different FOV were taken in each group. h Neurons expressing USP10 were lysed, and 0.1% Triton-100 soluble and insoluble fractions were extracted as recommended by the protocol. WB results showed the level of Tau5, pS199, and AT8 (short exposure or long exposure). 100–130kd is considered to be the high molecular weight (HMW) of aggregated Tau proteins. i, j Graphs showing the normalized protein levels of AT8 and pS199 in T-soluble fractions and T-insoluble fractions under the aforementioned conditions. k HMW total Tau and p-Tau of insoluble fractions were quantified. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
