Figure 3

A β1-42 promotes phosphorylation, cleavage and aggregation of tau by increasing GSK3β activity and by activating pro-caspase3. Sequential extraction was performed on Tau441-YFP-transfected cells treated with transfection reagent alone (control), 200 nM Aβ1-40 or Aβ1-42 for 24 h. Triton fraction was obtained by extracting intracellular tau with 1% Triton. Triton-insoluble fraction was then solubilized with 1% SDS to get SDS fraction. A) Triton fraction was electrophoresed and immunoblotted for total tau (HT7), p-tau(Ser^396/404) (PHF-1), p-tau(Thr²⁶²) (pS262), Tau421(tau-C3) and β-actin. β-Actin was measured from the same blots to ensure that equal protein was present in every lane. Aβ1-42 significantly induced p-tau at Thr²³¹ and increased levels of Tau421 were observed in Aβ1-42 treated cells, while no change occurred in Aβ1-40 treated cells when compared to cells without treatment. B) Quantification of western blots in (A). C) Representative images of western blots for SDS fraction with HT7, PHF-1 and pS262. All 3 forms of tau increased in Aβ1-42 treated cells. No change in tau level was found in Aβ1-40 treated cells. There was no detectable signal for tau-C3 in SDS fraction. D) Quantification of western blots in (C). E) Triton fraction was also blotted with anti-GSK3β antibody, anti-p-GSK3β (Y216) and anti-caspase-3 antibody. Aβ1-42 treatment resulted in an increase in phosphorylation of GSK3β, which represents higher GSK3β activity. More active caspase-3 was detected in Aβ1-42 treated cells. F) Quantification of western blots in (E). Histographs show mean +/− standard error, n =3. *p <0.05; ** p <0.01; ***p <0.005.