Hdac1

In vitro recombinant deacetylase activity was measured using the HDAC1 (BML-AK511), HDAC3 (BML-AK531), or HDAC6 (BML-AK516) FLUOR DE LYS fluorometric activity assay kits, per the manufacturer’s detailed protocol and instructions (Enzo Life Sciences). We assessed the inhibition of deacetylase activity with the following recombinant full-length 2N4R tau proteins: WT, P301L, L315R, S320F, and ΔR1–4 lacking the MTBR. All full-length tau proteins were added to a final concentration of 4.36 μM, while HDACs were preloaded at the following concentrations: HDAC1, 0.18 μM; HDAC3, 4.08 μM; HDAC6, 74.63 nM per the manufacturer’s protocol. Samples lacking tau but containing HDACs and substrate represent negative controls for HDAC inhibition (100% HDAC activity). Trichostatin A (TSA) was used as a positive control for inhibition of HDAC activity and was employed at a final concentration of 10 nM. Deacetylase reactions were carried out for 1 h at 30 °C, followed by the addition of developer for 45 min. Each individual HDAC assay was plotted as normalized HDAC activity relative to its respective TSA-treatment, which was established as a relative baseline representing maximal inhibition of HDAC activity. Fluorescence measurements were acquired using the FLUOstar Omega microplate reader (BMG LABTECH, Germany).