Pierce enhanced chemiluminescence ecl kit

Isolated histones (2 or 3 μg per sample) were run on a 15 % PAGE. Histones were transferred to Nitrocellulose (BioRad Laboratories, Hercules, CA, USA) or PVDF (Millipore, Billerica, MA, USA) membranes. Membranes were blocked in TBS-T with 5 % skim milk (DifcoLaboratories, Detroit, Mich, USA) for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies from Cell Signaling Technology, Danvers, MA, USA. The following antibodies were used: rabbit antibodies towards Histone H3 (1:1500), H3K9ac (1:1000) and H3K27ac (1:1000) and mouse antibodies to Histone H4 (1:1000). After washing, rabbit or mouse IgG-HRP secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated for 1 h at room temperature and then developed at Pierce Enhanced chemiluminescence (ECL) kit (Thermo Scientific, Rockford, IL, USA). The level of acetylated histone was quantified by Image J. All experiments were done in biological triplicate.

MEL cells were fractionated into cytoplasmic and nuclear components using a Cell Fractionation Kit—Standard (ab109719, Abcam), or total MEL cell protein lysates were generated. Proteins were detected via SDS-PAGE and Western blotting as described (Horos et al., 2012). Antibodies used were directed against Strap (sc-136083, Santa Cruz), Csde1 (NBP1-71915, Novus Biological), Stat5 (sc-835, Santa Cruz), Lamin B1 (ab133741, Abcam) and alpha Tubulin (ab4074, Abcam). Fluorescently labeled secondary antibodies for visualization with Odyssey were IRDye 680RD Donkey anti-Rabbit IgG (926–68073, Licor) and IRDye 800CW Donkey anti-Mouse IgG (925–32212, Licor), or using the Pierce enhanced chemiluminescence (ECL) kit (Thermofisher). Silver staining was performed using a SilverQuest™ Silver Staining Kit (LC6070, Thermofisher).