Hdac assay kit

For the direct comparison of HDAC1 vs. HDAC6 inhibition, HDAC activity was analyzed using the HDAC Assay Kit from Millipore (17–356). Briefly, recombinant HDAC1 or recombinant HDAC6 (both purchased from Epigenetek, Purity of HDAC1 is more than 60% and purity HDAC6 is more than 85%). was incubated with the fluorometric HDAC substrate according to the manufacturer’s protocol in the presence of DMSO vehicle or increase compound doses. In a secondary activator reaction, the fluorophore is only cleaved from the deacetylated substrate, allowing for quantification. Fluorescence was quantified on a FLUOstar-Optima or a FLUOstar Omega plate reader (BMG Biosciences). To ensure the compounds do not interfere with the detection system control experiments with standard deacetylated substrate were performed in the presence of high doses of all compounds.

For purification of HosA-TAP, six 1000 ml Erlenmeyer flasks containing 200 ml each of GXMM media were inoculated with A. nidulans conidia (5 × 10⁶/ml) and incubated with shaking at 37°C for 15 h. Affinity purification until the first elution by TEV protease was performed as described (Bayram et al., 2012 (link)). Aliquots of the elution were directly used for HDAC assays or frozen in liquid nitrogen for storage at -80°C.
Enzymatic activity of enriched HosA was measured in triplicates using either [³H] acetate-prelabeled chicken histones as substrate (Trojer et al., 2003 (link)) or fluorometric labeled peptides of a commercial (EMD Millipore) HDAC Assay Kit according to the manufacturer’s instructions. Briefly, 15 μl of the IgG eluate were mixed with HDAC Assay Buffer containing either Trichostatin A [TSA] in several concentrations (5, 20, 100, 250, 500, and 750 nM), or DMSO as control and subsequently was incubated for 60 min at 25°C. After addition of 20 μl activator solution, samples were further incubated for 15 min at 20°C. Fluorescence was measured using a FLUOstar Omega Plate Reader (BMG Labtech) set to 355 and 460 nm for excitation and emission, respectively. Before use, the instrument was tested and calibrated by creating a standard curve as described in the protocol of the HDAC Assay Kit.

Histone deacetylase activity was assessed using a fluorometric HDAC assay kit (EMD Millipore). For HDAC2-specific activity, immunoprecipitation with the specific antibody was performed before the assay as described previously (Guan et al., 2009 (link); Luo et al., 2014 (link)). Briefly, 0.5 μg of mouse anti-HDAC2 antibody (Abcam) was added to the cellular lysates and incubated for 12 h at 4°C. Then 20 μl of protein G-Agarose (Sigma-Aldrich) was added and incubated on a tube rotator for 4 h at 4°C. Beads were centrifuged at 2500 × g and washed five times in immunoprecipitation buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, pH 8.0, supplemented with 1 mM PMSF). Finally, HDAC assay substrate was added to the beads and incubated at 30°C for 45 min, after which the reaction was stopped and fluorescence was measured according to the instructions of HDAC assay kit.