Journal: Molecular and Cellular Biology
Article Title: Runx2 Is a Common Target of Transforming Growth Factor ?1 and Bone Morphogenetic Protein 2, and Cooperation between Runx2 and Smad5 Induces Osteoblast-Specific Gene Expression in the Pluripotent Mesenchymal Precursor Cell Line C2C12
Figure Lengend Snippet: ) and three mutant DNAs, M1 (one mismatch in the putative Runx binding site [CACCACA]), M2 (a perfect match [GACCACA]), and M3 (putative Smad binding site [CAGACA]). Putative Runx2 and Smad binding sites are indicated by solid and dotted underlining, respectively, and mutated nucleotides are marked by asterisks. (B) EMSA was performed by using the TβRE probe and nuclear lysates obtained from C2C12 cells untreated or treated with TGF-β1 or BMP-2 for 24 h. The reactions were performed in the presence or absence of PEBP2β/Cbfβ protein. A 50-fold molar excess of unlabeled mutant oligonucleotide was incubated with the nuclear lysates as competitor DNA (Ct). The arrow and arrowhead indicate the TβRE binding complex and free probe, respectively. (C) EMSA was performed by using the same nuclear lysates and TβRE probes in the presence or absence of a polyclonal antibody which recognized all members of the Runx α subunit (anti-α; lane 2 and 5) or β subunit (anti-β; lane 3 and 6) or a monoclonal antibody which specifically recognized Runx2 (anti-Runx2; lanes 8, 10, and 12). The arrowheads and arrows indicate the positions of Runx2 and Runx-antibody complexes, respectively. (D) Time course induction of Runx2 was analyzed by EMSA using nuclear lysates prepared from cells treated with TGF-β1 for 0, 4, 12, 18, 24, 72, and 120 h. A nuclear extract prepared from cells cultured for 72 h in the absence of TGF-β1 was used as a control. EMSA was performed with the TβRE probe in the presence or absence of a 50-fold molar excess of unlabeled M3 as competitor. The arrow indicates the TβRE binding complex. Ct, competitor.
Article Snippet: The mouse pluripotent mesenchymal precursor cell line C2C12 was purchased from the American Type Culture Collection C2C12 and MC3T3-E1 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), and penicillin G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 in air.
Techniques: Mutagenesis, Binding Assay, Incubation, Cell Culture