T6793 monoclonal

Cells were washed with phosphate-buffered saline (PBS) and collected using the EpiQuik Total Histone Extraction Kit (EpiGentek). Protein concentrations were determined using the micro BCA kit (Thermo-Fisher Scientific) according to manufacturer’s protocol. Protein samples were supplemented with reducing sample buffer (Thermo Scientific) and denaturized by incubating at 95 °C for 5 min. The nonspecific binding was blocked by incubation of the PVDF membrane in 5% bovine serum albumin (BSA), diluted in Tris Buffered Saline Tween (TBST, 50 mM TRIS, 150 mM NaCl, 0.1% Tween-20 (Applichem) for 1 h at room, temperature followed by incubation with primary antibodies overnight. The antibodies, diluted in TBST, were directed against α-tubulin (1:5000, T6199, Sigma-Aldrich), acetylated α-tubulin (1:5000, T6793 monoclonal, Sigma-Aldrich), histone H3 acetyl k9+k14 (1:1000, 9677L, Cell Signaling), and histone 4 (1:1000, ab10158, Abcam). Secondary antibodies conjugated with horseradish peroxidase (Dako, 1:5000, 1 h, RT) where used prior to detection with ECL substrate (life technologies) with a LAS 4000 Image Analyzer (GE Healthcare). Luminescent signals were quantified with ImageQuant TL 7.0 software (GE Healthcare) and then graphed with GraphPad Prism software. A mild reblotting buffer (Merck Millipore Corp.) was applied to strip the blots.