Epiquik global histone h3 acetylation assay kit

Chromatin immunoprecipitation (CHIP) was performed using the EpiQuik™ Global Histone H3 Acetylation Assay Kit (Epigentek, USA) according to the manufacturer’s instructions. In brief, cross-linked chromatin DNA was sonicated into 200–1000-bp fragments. This fragmented chromatin was immunoprecipitated using an anti-acetyl-histone H3. Normal mouse IgG was used as the negative control. qRT-PCR was performed to assess the expression of CD133 promoter.

Global histone H3 acetylation levels in resistant and susceptible larvae were determined using the EpiQuik™ global histone H3 acetylation assay kit (EpigenTek, USA). Samples were extracted in three volumes of buffer with glycerol on ice for 5 min, and the supernatant was mixed with 100% trichloroacetic acid and incubated on ice for 30 min before centrifugation (10 min, 13,523 x g, 4 °C). The pellet was washed twice with acetone and dissolved in water. The histone protein concentration was estimated using the bicinchoninic acid (BCA) method and the extract was divided into aliquots for storage at −80 °C prior to analysis. Following extraction, the histone proteins (1–2 μg) were coated onto the strip wells and acetylated histone H3 was detected using a high-affinity antibody. The ratio in resistant and susceptible larvae was estimated using a horseradish peroxidase (HRP)-conjugated secondary antibody and the colorimetric signal was quantified by measuring the absorbance at 450 nm.

Global H3 acetylation was determined using the EpiQuik™ Global Histone H3 Acetylation Assay Kit (Epigentek) following manufactures protocols. Protein concentration of the nuclear extracts was quantified using a BCA assay, and 1.5 μg of total protein was used from each experimental group. Samples were run using three biological replicates in technical triplicate and final absorbance readings were subjected to a Welch two subject t-test to establish statistical significance for each comparison. Acetylation levels were displayed as a ratio compared to the corresponding control H3 acetylation abundance.

The effects of TNFα alone or in combination with HCA on H3 and H4 histone acetylation were evaluated with the EpiQuik™ Global Histone H3 Acetylation Assay Kit and EpiQuik™ Global Histone H4 Acetylation Assay Kit (Epigentek, Farmingdale, NY, USA). Following the manufacturer’s instructions, histones were extracted from HH that had been treated for 24 h with 5 ng/mL TNFα alone or combined with 500 μM HCA. The histones were spotted onto the wells, and then an antibody specific for acetylated histone H3 or H4 was added. After washing the wells, HRP-conjugated secondary antibody was added, followed by the detection reagent, and absorbance was measured with GloMax® Discover Microplate Reader at 450 nm. Percent acetylation was calculated as OD (treated sample − blank)/OD (untreated control − blank) × 100%.

Midguts were dissected from male and female third-instar larvae (F0, F1), flash frozen in liquid nitrogen, homogenized and stored at −80°C. Global levels of lysine-specific histone H3 acetylation were determined using the EpiQuik Global Histone H3 Acetylation Assay Kit (Epigentek Group Inc.) according to the manufacturer’s protocol. Fold changes of relative histone acetylation were calculated for treatment groups exposed to bacteria against the corresponding control groups.