Epigenase hdac activity inhibition direct assay kit

In vitro HDAC activity was measured using the Epigenase HDAC Activity/Inhibition Direct Assay Kit (Fluorimetric, Epigentek; catalog no. P-4035) according to the manufacturer's instructions. Briefly, the full-length coding sequence of LcHDA2, LcHDA6 and LcSRT2, were cloned into pET32a(+) (Novagen) and transformed into Escherichia coli strain BL21(DE3). The gene-specific primer pairs are listed in Table S1. The recombinant proteins were affinity purified using His60 Ni Superflow Resin (TransGen, Beijing). Then, 5 μg of purified proteins per well was incubated with 50 ng of substrate for 90 min at room temperature. The HDAC-deacetylated products can be recognized with a specific antibody. The ratio or amount of deacetylated products, which is proportional to the enzyme activity, can then be fluorometrically measured by reading the fluorescence in a fluorescent microplate reader (FLx800, BioTek) at 355_ex/460_em and data acquired using Gen5™ Analysis Software 2.06.10. The HDAC inhibitor TSA was used to demonstrate the specificity of deacetylation activities. That the activity of the HDAC enzyme is proportional to the OD intensity was calculated with formula: HDAC Activity (RFU/min/μg) = (Sample RFU − Blank RFU)/(Protein Amount (μg)^* × min^**).

HSV-2–infected or romidepsin-treated cells were fractionated with Minute Cytoplasmic & Nuclear Extraction Kits (SC-003, Invent Biotechnologies Inc.), and at least 0.5 μg of nuclear extract was used to determine HDAC activity (Epigenase HDAC Activity/Inhibition Direct Assay Kit, P-4034-96, EpigenTek). Absorbance was measured using a SpectraMax M5e microplate reader and SoftMax Pro 7.1 GxP software (Molecular Devices).

The frozen colon tissue was weighed and then homogenized on the ice surface. Homogenate was obtained by centrifugation at 1000 g and 4°C for 20 min. TNF-α and IL-6 levels of colonic homogenate were determined using ELISA kits (Beijing Cheng Lin Biological Technology Co., LTD., Beijing, China) according to the manufacturer’s protocol. The HDAC activity in the nuclear extract of colon tissue was measured using the Epigenase HDAC Activity/Inhibition Direct Assay Kit (Epigentek Group Inc., United States) and following the manufacturer’s instructions. Moreover, the binding capacity of nuclear P65 to the NF-κB consensus site in the nuclear extract of colon tissue was evaluated by measuring the activated NF-κB P65 using the ELISA-based Trans-AM^TM NF-κB P65 Kit (Active Motif, Carlsbad, United States); this evaluation was performed according to the manufacturer’ instructions by referencing the similar study (Cichocki et al., 2014 (link)). The results were expressed as absorbance (OD_450nm/mg protein).

Matrigel was dissolved in ice-cold Cell Recovery solution (Corning), and nuclear protein extracts were prepared from recovered wild-type, Hdac1- and Hdac2-deficient enteroids grown for 5 days, by using Abcam nuclear extraction kit (ab113474). 7.5 µg of nuclear extracts were used to measure nuclear HDAC activity with the colorimetric Epigenase HDAC activity/inhibition direct assay kit (Epigentek), on a Versamax ELISA microplate reader at 450 nm (Molecular Devices), according to the manufacturer’s protocol (n = 3; 2–3 wells for each). As a control, nuclear extracts were incubated with the HDAC pan-inhibitor Trichostatin A (1 μM). Results are expressed as the mean ± SD. Statistical significance was determined by Student’s t-test.

On the day of the assay cell fractioning was performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents. Cytosolic extracts were processed with Epigenase HDAC Activity/Inhibition Direct Assay Kit (Epigentek) following the manufacturer's instructions. Colorimetric reaction was detected at 450nm using ELx800 Absorbance Microplate Reader (BioTek).