FIGURE 5.

Expression of wild-type and mutant RUNX2 proteins in reconstituted null cells is sustained throughout the osteoblast differentiation time course. A, schematic illustration of the reconstitution biological assay. Three days after plating, Runx2 null cells were infected with adenovirus expressing wild-type or mutant RUNX2 protein for 4 h. Cells were then cultured for additional 12 days in osteo-inductive mixture and in the presence and absence of 100 ng/ml BMP2. B, Runx2 null cells were infected with wild-type (50 m.o.i.), Y428A (70 m.o.i.), HTY (60 m.o.i.), and Δ391 (37 m.o.i.) and cultured for 12 days in 6-well dishes. Cells at indicated days were directly lysed in plates as described under “Experimental Procedures.” For comparison of RUNX2 expression levels, equal amounts of cell lysates were resolved on 10% SDS-PAGE for Western blot analysis. Blots were probed with monoclonal RUNX2 antibody, stripped, reprobed with LaminB antigen, and used as loading control.