Epiquik total histone extraction htkit

Caco2 cells were cultured in 100-mm Petri dishes (Sarstedt) in DMEM supplemented with 10% FBS and 1-mM sodium pyruvate (all purchased from Wisent), at 37°C in a 5% CO₂ atmosphere-humidified incubator. Transfections were carried out as cells were plated out in 6-well plates (Sarstedt). Approximately ∼1.2 × 10⁶ cells per well were plated and concomitantly transfected with 20-nM ASO and 4.5-μl Lipofectamine 3000 (Life technologies). After 24 h, cells were washed and scraped in ice-cold PBS. One fifth of the cells was used for RNA extraction (see the Quantitative PCR analyses section) and the rest for histone extraction with the EpiQuik™ Total Histone Extraction HTKit (Epigentek) according to manufacturer's protocol. Approximately 1 μg of total histones was fractionated through 12% acrylamide SDS-PAGE. Macroh2a1 polyclonal rabbit antibody (Active motif no39594) diluted 1000X was used for western blot analysis. Histone 3 monoclonal antibody (Active motif no61476) diluted 1000X was used as a loading control. Results are from three independent experiments, each performed in triplicate.

To determine the ratio of H3 K9me2/H3 total N2 (WT) and CL2006 animals were incubated in liquid culture with vehicle (DMSO 1 %), or 0.1 μM of G9a inhibitor in a 96‐well plate format as already mentioned. For chronic treatment, four‐day‐old animals were collected with M9 buffer. Histone extraction was performed following the manufacturer's instructions (EpiQuik Total Histone Extraction HT Kit, EpiGentek, #OP‐0007‐192). The samples were resolved in a 14 % SDS‐gel, as previously described.^[12] To capture chemiluminescence signals were used Amersham Imager 680 and Western blot quantifications were performed using ImageLab software (Bio‐Rad). Immunoblots were probed with anti‐H3 K9me2 (1 : 1000) (Epigentek, #A‐4035), and anti‐H3 total (1 : 1000) (Cell signaling, #9715).