Anti acetyl histone h4 lys12

Western blot was done according to previously described (24 (link)). Briefly, yeast cells were fixed and lysed in 20% trichloroacetic acid (TCA) using glass beads. The resultant pellet was extracted with Laemmli buffer and denatured in boiling water for 5 min, then the proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membranes (Millipore, Cat# HATF00010). The mouse monoclonal anti-HA (Sigma, Cat# 11867423001), mouse monoclonal anti-PGK1 (Abcam, Cat# ab113687), rabbit polyclonal anti-H3K4me3 (active motif, Cat# 39159), rabbit polyclonal anti-Histone H4-C terminal (Affinity, Cat# DF6950), rabbit polyclonal anti-Acetyl-Histone H4(Lys8) (Affinity, Cat# AF4353), rabbit monoclonal anti-Acetyl-Histone H4(Lys12) (Cell Signaling Technology, Cat# 13944S), and rabbit polyclonal anti-Acetyl-Histone H4(Lys16) (Affinity, Cat# AF3636) antibodies were used for western blot. Quantification was performed using ImageJ software.

After indicated doses of treatment, IK cells were maintained at 37°C and 5%CO2 until fixation at 24 hours post-treatment. Cells were washed twice with phosphate-buffered saline (PBS), fixed for 20 min with 4% paraformaldehyde, permeabilized with methanol during 5 min, washed and maintained with PBS. After blocking (5% HS +5% FBS +0,2% Glicina +0,1% Tritó in PBS) during 1 h, cells were incubated overnight at 4°C with a monoclonal anti-γH2AX antibody (monoclonal, Millipore), anti-Acetyl-Histone H4 (Lys12) (polyclonal, Cell-Signal) 1∶500 in PBS, and washed and incubated with FITClabeled secondary antibody (Sigma) 1∶500 in PBS in the dark for 1 h at room temperature. Cells were then washed, counterstained, and stained with bis-benzimide fluorescent dye (Hoechst 33258), to a final concentration of 0.5 mg/ml, in the dark for 35 minutes. Cells were counted under epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). Aleatory sampling methods were used to select the images and all the cells in each selected image were screened. The γH2AX foci per nucleus were counted by eye. Cells were judged as “positive” for γH2AX foci if they displayed 10 or more discrete dots of brightness. For quantitation of foci, a minimum of 300 cells were analysed for each time point.