Fluor de lys hdac fluorometric activity assay kit

The In Situ Histone Deacetylase (HDAC) Activity Fluorometric Assay Kit (Sigma-Aldrich) was used to measure HDAC activity according to the manufacturer’s instructions. The HDAC substrate was added to wells containing live cells directly to the treatment media after 23 h treatment. For HDAC activity assays on the nuclear extracts, enzyme substrate was added concurrently with statin treatments in phosphate-buffered saline. After 1-h incubation, the HDAC developer was added and the plate was incubated for a further 30 min and fluorescence read with an excitation wavelength of 368 nm and an emission wavelength of 442 nm.
Confirmatory experiments were carried out with the Fluor De Lys^® HDAC fluorometric activity assay kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instructions. HeLa nuclear extract provided with the kit and HepG2 nuclear extract prepared as above were incubated with treatments and substrate for 30 min. Following incubation with developer for 10 min, fluorescence was read with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

For immunoprecipitation, hippocampal tissues were dounce homogenized in IP buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% Triton X-100, 0.1% NP-40). Lysates were pre-cleared with 30 μL of Protein A Sepharose beads (Sigma-Aldrich) at 4 °C for 30 min and incubated with 5 μg of primary antibody overnight on a tube rotator at 4 °C. Beads were washed three times in washing buffer (20 mM HEPES KOH adjust pH 7.9, 0.1 M KCl, 0.01% NP-40) and boiled in 2× loading buffer for western blot analysis. For HDAC1 and p300 activity assay, hippocampal tissues were lysed in IP buffer and immunoprecipitated with anti-HDAC1 antibody (Abcam, ab7028) or anti-p300 antibody (Thermo Fisher, MA1-16608). The washed beads with bound HDAC1 or p300 were assessed for HDAC or histone acetylase activity using the FLUOR DE LYS® HDAC Fluorometric Activity Assay Kit (Enzo Life Sciences) or HAT Activity Assay Kit (Abcam, ab65352) according to the manufacturer’s instructions. HDAC1 or p300 activity was normalized to input HDAC1 or p300 protein levels.