Anti histone h3 acetyl k9 antibody

Protein samples were mixed with Laemmli buffer (BioRad) and β-mercaptoethanol (Sigma). Each sample was analyzed in triplicate. Next, samples were denatured, loaded on a gel (0.1 µg protein per sample), and separated by electrophoresis (60 min, 125 V). Then proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked with 3% BSA for 1 h at RT and incubated with primary antibodies (anti-histone H3 (tri methyl K9) antibody (Abcam), anti-histone H4 (tri methyl K20) antibody (Abcam), anti-histone H4 (acetyl K8) antibody (Abcam), anti-histone H3 (tri methyl K4) antibody (Abcam), anti-histone H3 (tri methyl K27) antibody (Abcam), and anti-histone H3 (acetyl K9) antibody (Abcam)) in TBST for 16 h at 4 °C. The membranes were then washed three times for 10 min with TBST, and were incubated with secondary antibody (anti-rabbit) for 1 h at RT and washed. The chemiluminescence reaction was performed with Clarity Western ECL Substrate (BioRad). Signals were visualized with ChemiDoc Imaging System (BioRad), and signal intensities were measured using ImageLab software (BioRad).

Nuclear and cytoplasmic protein extraction was performed using a nuclear and cytoplasmic protein extraction kit (Cat# MP1551-100T, MKBio, China), according to the manufacturer’s instructions. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA). Equal amounts of nuclear protein for each sample (30 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunodetection was performed using an enhanced chemiluminescence ECL system (Cat# 36222ES60, Yeason, China). Histone H3 served as the loading control. The primary antibodies used in this study included anti-histone H3 antibody (1:1000, Cat# ab1791, Abcam, UK), anti-cyclin D1 (1:200, Cat# ab16663, Abcam, UK), HDAC3 antibody (1:1000, Cat# AF5349-50 μL, Affinity, China), anti-histone H3 (acetyl K9) antibody (1:500, Cat# ab32129, Abcam, UK), and FoxO3a (75D8) rabbit mAb (1:1000, Cat# 2497S, Cell Signaling Technology, USA). The secondary antibodies were goat anti-rabbit IgG HRP-linked antibody (1:2000, Cat# 7074S, Cell Signaling Technology, USA) and anti-mouse IgG HRP-linked antibody (1:2000, Cat# 7076S, Cell Signaling Technology, USA). The relative optical density of the protein bands was measured using the ImageJ software, and the protein levels were normalized to histone H3.