Anti acetyl histone h3 lys9

After treatments, 4T07 cells were solubilized in Lysis Buffer (20 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 3,5 mM SDS, 13 mM deoxycholic acid) and proteins extracted were separated on a 4–15% Tris/Glycine/SDS gel (BIO RAD). A standard protocol was used for transfer and blotting. Anti-acetylated tubulin antibody was purchased from Sigma-Aldrich (#T7451). Anti-H3 antibody was clone96C10 (Cell Signaling Technology (CST); cat # 3638). Anti-acetyl-histone H3(Lys9) was 07–352 (Merck Millipore), and anti-acetyl-histone H3 (Lys27) was #8173 (CST). Anti-GAPDH antibody was purchased from Abcam (#ab8245) and the anti-mouse IgG HRP-linked secondary antibody was purchased from Cell Signaling Technology and were used at recommended concentrations. Signal was detected using Clarity ECL chemiluminescent substrates (Bio-Rad) and ChemiDoc Imaging System from the same company was used to quantify the intensity of bands. Changes in protein levels were quantified relative to control using Image Lab software (Version 6.0.1) after normalization to GAPDH. Western-blot images presented are three biological replicates.

Brain cortex homogenates from groups 1–6 (see Experimental groups of animals) were prepared as described in our previous paper [31 (link)] and immunodetection was assessed according to Pilchová et al. [71 (link)]. Membranes were probed with antibodies used for immunofluorescence, rabbit polyclonal Anti-acetyl-Histone H3 (Lys9) (Merck Millipore, Darmstadt, Germany, 1:500) and Anti-acetyl-Histone H4 (Lys12) (Merck Millipore, Darmstadt, Germany, 1:500) with secondary goat anti-rabbit IgG HRP-conjugated antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, 1:5000). Immunoblots were visualized by ECL chemiluminescent substrate (ThermoFisher Scientific, Dreieich, Germany) on Molecular Imager Gel Doc XR System (Bio-Rad, Hercules, CA, USA) and bands of interest were analyzed by Quantity One (Bio-Rad, Hercules, CA, USA). All assays were performed in triplicates.

Total cellular extracts were separated in a 12.5% polyacrylamide gel (SDS-PAGE). Following electrophoresis, proteins were transferred onto a nitrocellulose membrane at 60 mA for 90 minutes, using a semidry system (TE77X, Hoefer, USA). Membranes were blocked for 1 h in 5% nonfat dry milk and diluted in PBS-T (PBS containing 0.05% (v/v) Tween-20), under low agitation. Subsequently, for phosphorylated H2A, H3 acetylation, and Pgk1p detection, membranes were incubated overnight at 4°C with the primary polyclonal antibodies anti-γH2AX (1 : 5000, Abcam), anti-acetyl-Histone H3 (Lys9) (1 : 5000, Millipore), or anti-PGK1 (1 : 5000, Molecular Probes), respectively. The next day, membranes were washed 3 × 5 minutes with PBS-T, followed by a 1 h room temperature incubation with the secondary antibodies anti-rabbit (for γH2AX and acetyl-Histone H3) or anti-mouse (for Pgk1p) from Jackson Laboratories. Chemiluminescence detection was performed using the Immobilon ECL detection system (Millipore-Merck) and a Chemi-DOC XRS system (BioRad) or X-ray ortho CP-G films in an X-ray film processor (Curix 60, Agfa Healthcare).