Fig. 6: PADI2-catalyzed citrullination of RUNX2 is required for RUNX2 stabilization.

A MC3T3-E1 cells were transfected with empty vector (EV), Strep-Runx2 wild type (Wt), or R-to-K mutants and cultured in osteogenic media for 3 days after transfection. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified using ImageJ software and normalized with β-Actin. The arrow indicates non-specific bands (n.s). Western blot data were collected from three independent experiments; the representative results are shown here. B, C pOB cells and hMSCs were transfected with EV, Strep-Runx2 WT, or R381K mutant plasmids and cultured in osteogenic media for 2 days. Cells were lysed and western blot analysis was performed. β-Actin was used as a loading control. Strep-RUNX2 level was quantified and normalized with β-Actin using ImageJ software. Western blot data were collected from two independent experiments; the representative results are shown here. D 293 T cells were transfected with Strep-Runx2 Wt or R-to-K mutants with or without Myc-Cbfβ plasmids and then incubated for 3 days after transfection. Cells were lysed, immunoprecipitated with Strep-Tag II magnetic beads, and immunoblotted with indicated antibodies. Co-IP experiment was performed in three independent experiments; the representative results are shown here. E MC3T3-E1 cells were transfected with Strep-Runx2 Wt or R-to-K mutants and cultured for 2 days after transfection. Cells were fixed with 4% PFA, permeabilized, and then immunofluorescent staining was performed using anti-Strep-Tag II antibody. DAPI was used for the nucleus. Three independent experiments were performed and the representative results are shown here. Scale bar, 20 μm.