Western Blot Analysis of Bone Markers

Cells were harvested and then lysed with RIPA lysis buffer containing 1.0 mM protease inhibitor cocktail, 1.0 nM DTT and PMSF for 30 min at 4 °C. Then, the protein supernatant was collected using a microcentrifuge at 13,000 rpm for 15 min at 4 °C. Appropriate protein (50 μg) of samples were subjected to 10–12% SDS-PAGE gel electrophoresis and electroblotted into PVDF membranes (Millipore, Darmstadt, Germany) as previously described^{54 (link)}. The membranes were blocked with skimmed milk solution (5%) and incubated with the following primary antibodies: GAPDH (1:3000; Origene, USA), β-Actin (1:3000; Abclonal, USA), Osteocalcin (1:1000; Santa Cruz Biotechnology, USA), RUNX2 (1:1000; Proteintech, China), SP7/Osterix (1:1000; Abcam, USA), BARD1 (1:500; Santa Cruz Biotechnology, USA), FBXO25 (1:300; Santa Cruz Biotechnology, USA), CUL3 (1:500; Santa Cruz Biotechnology, USA), H2A (1:1000; Absin, China), H2B (1:1000; Abcam, USA), H3 (1:1000; CST, USA), H2AK119ub (1:1000; CST, USA), H2BK120ub (1:1000; CST, USA), and H3K4me3 (1:2000; CST, USA). The results were visualized using a MiniChemi 610 System Instrument (Beijing, China).