(I, J) Only at 125 mM of trehalose, both phosphorylated TAU (P<0,001, I) and total TAU levels (P<0,001, J) are decreased. (G, H) AlphaLISA results reveal that trehalose significantly reduces hTAU10 (P<0,001) and AT8 TAU aggregation levels versus control (P = 0,006 at 31,5 mM and P = 0,014 at 125 mM). (F) CellTiter-Glo® results show that trehalose is highly toxic at 250 mM (P<0,001). (D, E) Also general TAU phosphorylation is reduced (AT8/hTAU10 P<0,001 D) to a similar extent as the reduction in total TAU (HT7/hTAU10 P<0,001 E). (B, C) Rapamycin dose-dependently reduces hTAU10 (P<0,001 B) and AT8 aggregated TAU measured with AlphaLISA (P = 0,025 at 10 nM and P<0.001 at 1 μM C) and compared to DMSO. (A) CellTiter-Glo® data showing that rapamycin is not toxic (P = NS). Thermo Scientific PageRuler Plus Prestained Protein Ladder is a mixture of nine (9) blue-, orange- and green-stained proteins (10 to 250kDa) for use as size standards in protein electrophoresis (SDS-PAGE) and Western blotting.Features of Pa. Model validation: autophagy inducers reduce TAU hyperphosphorylation and aggregation in TAU-P301L neurons. (I) Immunostaining for AT8 and neuronal HuC/D after 1%Triton/PFA fixation, to remove monomeric TAU, reveals AT8-positive neurons after K18 seeding. Note the presence of monomeric 3R and 4R TAU protein in the soluble fraction. Aggregates are only present in the insoluble pellet after addition of 6nM or 50nM of K18 fibrils. (G, H) Representative Western blots after Sarkosyl extraction showing soluble (S) and insoluble (IS) fractions after blotting with antibodies against total TAU (HT7 G) and hyperphosphorylated TAU (AT8 H). Thermo Scientific Spectra Multicolor Broad Range Protein Ladder is a 4-color protein standard containing 10 prestained proteins (10 to 260kDa) for use in gel electrophoresis and Western blotting. 1236kDa) in K18-seeded samples suggest the presence of TAU aggregates. SuperSignal West Femto Maximum Sensitivity Substrate.
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