Hdac4

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).

Concisely, cells and tissues were incubated with Protein Extraction Reagent (Phygene, Fuzhou, China) containing Protease Inhibitor Cocktail for 20 min on ice, and total proteins were extracted by centrifugation at 10,000g for 20 min. Then, 20 μg of protein were loaded onto 8%-12% bis–tris-acrylamide gels (Phygene). The proteins were wet-transferred onto nitrocellulose membranes, which were then blocked with 5% defatted dry milk. Next, the primary antibodies specific to BCL2-associated x protein (Bax; 1:1000; Thermo Fisher), cleaved caspase 3 (1:1500; Thermo Fisher), B-cell lymphoma-2 (Bcl 2; 1:50; Thermo Fisher), HDAC4 (1:1500; Thermo Fisher) and GAPDH (1:1000; Thermo Fisher) were used to incubate the membranes. After being probed with secondary antibodies (Thermo Fisher), the membranes were analyzed under a Bio-Rad image analysis system with the use of eyoECL Plus (Beyotime).