Anti h3k9 k14ac

Total proteins were extracted using extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 0.5 mM PMSF, 10% glycerol, 10 mM DTT and protease inhibitor cocktail). The protein samples were separated by 12% SDS-PAGE gels. After separation by electrophoresis, proteins were transferred onto the PVDF membrane and detected using the primary antibodies anti-H3K9K14ac (Millipore, Cat No. 06-599), anti-H3K4me³ (AbCam Cat No. ab8580), and anti-Actin (Abbkine Cat No. A01050). Quantification of the band intensities on the western blot was performed using the Image J software.

For chromatin immunoprecipitation (ChIP) assays, purified pro-B cells from the bone marrow of Hdac7^+/− and Hdac7^fl/− mice were crosslinked for 15 min in 1% formaldehyde, followed by inactivation in 125 mM glycine for 5 min and by two washes in cold PBS. Afterward, samples were incubated in cell lysis buffer from LowCell# ChIP kit (Diagenode) for 30 min at 8°C and sonicated with M220-Focused Ultra Sonicator (Covaris) according to manufacturer's instructions. Next steps of ChIP experiments were performed using resources from the LowCell# ChIP kit (Diagenode) according to the manufacturer's instructions. The following antibodies were used for immunoprecipitation: anti-HDAC7 (Abcam, HDAC7-97, 2.5 μg), anti-H3K9/K14ac (Millipore, 06-599 2.5 μg), anti-H3K27me3 (Millipore, 07-449, 2.5 μg), anti-H3K27Ac (Abcam, ab4729, 2.5 μg) and anti-H3k9me3 (Abcam, ab8898, 2.5 μg). Real-time quantitative PCR (RT-qPCR) was performed in triplicate and the results analyzed. Data are presented as the ratio between the HDAC7-bound fraction and histone modification antibody relative to the input control. ChIP-qPCR primer pairs are shown in supplemental information Supplementary Table S1.