Figure 3
The teleost Runx2b is functional in mammalian osteoblasts and interacts with the 1α,25-dihydroxyvitamin D3 pathway. (A) ROS17/2.8 osteoblasts were co-transfected for 12 hours with a Myc-tagged version of the Danio Runx2b homologue and the promoter of the rat osteocalcin driving the expression of the luciferase reporter gene. Cells were subsequently incubated for 18 hours in the absence (white bars) or in the presence (grey bars) of 1α,25-dihydroxyvitamin D3 (Vitamin D) before being assayed for relative Luciferase activity. (B) Western blot performed on nuclear extracts from ROS17/2.8 cells transfected or not with the Danio Runx2b homologue. (C) Nuclear extracts (150 μg) from ROS 17/2.8 cells cultured in the presence of 10-8 M 1α,25-dihydroxyvitamin D3 for 18 h were incubated with GST or with the indicated GST fusion proteins (1.5 μg) previously bound to 20 μl of glutathione-Sepharose beads. Precipitated VDR (upper panel) and GST-Runx (lower panel) proteins were then detected by Western blotting. The migration of molecular weight standards is indicated on the left.