Tht dye

GST-Htt(Q)₂₅, GST-Htt(Q)₄₃ and FKBP12 proteins were purified and determined the concentrations and purity by SDS-PAGE staining with Coomassie blue and Bradford assay, respectively (details in Supplementary Materials and Methods). The amyloid properties of HTT proteins harboring different polyglutamine expansions were monitored using the Thioflavin T (ThT) fluorescence assay, as described previously29 (link). GST-Htt(Q)_n (n = 25 or 43) at a concentration of 3 μM was treated with thrombin, and incubated in the presence or absence of 3 μM FKBP12 for 1 or 7 days. At the indicated time, ThT dye (Sigma) was applied to a final concentration of 100 μM. Fluorescence measurements were obtained at an excitation wavelength of 442 nm, and the emission spectra from 460 to 600 nm were recorded with a Hitachi F4500 Fluorescence Spectrometer. For determining the amyloid properties of the sorted materials from the P1 fraction, 4 × 10⁶ particles were collected and centrifuged at 14 K rpm for 30 min at 4 °C. Pellets were resuspended with 80 μl ddH₂O and then stained with ThT dye; the resulting fluorescence spectra were measured as described above. Values are shown as means ± Standard Error of the Mean (SEM) calculated from three independent experiments.

ThT dye (Sigma-Aldrich, Co) was dissolved in Milli Q water and filtered through a 0.2 µm syringe filter to obtain a stock concentration of 2.3 mM. The ThT dye was diluted to give a final working concentration of 200 µM. Aggregate-free Aβ(1–40) solution at a stock concentration of 250 µM was prepared by dissolving the synthetic Aβ(1–40) powder (ChinaPeptides Co., Ltd) in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP). The HFIP in the mixture was left to evaporate overnight to obtain the dry powder. About 20 ul of 100 mM NaOH was added to the HFIP treated monomeric Aβ peptide powder to further dissolve the Aβ(1–40) and remove any pre-formed aggregates. After centrifuging at 14,000 rpm for 5 min, the supernatant was collected and 50 mM of sodium phosphate buffer was added to obtain a final working concentration of 50 µM monomeric solution of Aβ(1–40) peptide. The fluorescence intensity of ThT was measured using Cytation 5 cell imaging multi-mode (Biotek Instrument Inc., USA) reader with λ_ex = 430 nm and λ_em = 480 nm. The kinetics experiment was run for 72 h at 37 °C to monitor the formation of the Aβ(1–40) fibrils from the monomeric Aβ(1–40) peptide solution.