Acetylated h3k9 k14

Histone preparations were made from young and aged mice BMSCs. Equal amounts of core histones were resolved by 15% SDS-PAGE, transferred onto PVDF membranes and probed with the following antibodies: acetylated H3K9/K14 (#9677, Cell Signaling Technology, Danvers, MA, USA), acetylated H4K12 (#13944, Cell Signaling Technology, Danvers, MA, USA), HDAC1 (#34589, Cell Signaling Technology, Danvers, MA, USA), HDAC3 (#85057, Cell Signaling Technology, Danvers, MA, USA), HDAC4 (MA5-15580, ThermoFisher scientific), HDAC5 (#98329, Cell Signaling Technology, Danvers, MA, USA), HDAC6 (PA1-41056, ThermoFisher scientific), total H3 (#4499, Cell Signaling Technology, Danvers, MA, USA), total H4 (#13919, Cell Signaling Technology, Danvers, MA, USA) and β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA).

For each ChIP, 5 × 10⁶ HEK293T cells transfected with pCCLteteGFP, pCCLteteGFP-2A-HAELF2A, and pCCLteteGFP-2A-HAELF2B were cross-linked with 1% (w/v) formaldehyde for 10 min and were quenched with 1 M glycine to a final concentration of 20 mM. Nuclear lysates were sonicated for 25 cycles, 30 s on, 30 s off using a Bioruptor sonicator (Diagenode). Antibodies for immunoprecipitating protein/DNA complexes include: acetylated H3K9/K14 (#9677, Cell Signaling); CTCF (07-729, Millipore); and HA (ab9110, Abcam). Protein G-conjugated agarose beads (Millipore) were used to immunoprecipitate antibody-bound chromatin complexes, and all subsequent steps were performed according to the manufacturers’ instructions. After de-crosslinking, phenol/chloroform extraction, and ethanol precipitation, PCR was performed on genomic DNA targets using Phusion polymerase with GC buffer (Finnzyme). Primer sequences are in Additional file 1: Table S1.