Cul 3

Whole-cell lysates were separated using SDS-PAGE and transferred onto PVDF membranes. The proteins were detected with HSC70 (sc7298), HSP60 (sc-13115), CUL-3 (sc-166110), NFATc1 (sc-7294), Cyclin D1 (sc-8396), and Ub (sc-8017) (Santa Cruz Biotechnology), FLAG (F1804, Merck), and β-actin antibody (G043, ABM). The blots were cropped and the full image of the non-crop blots were presented in Supplementary Figures.

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).