Figure 6.

Recruitment of Smads to sites of active transcription is coupled with the regulation of gene expression. HeLa cells were transfected with 0.45 95 g of pTβRE-Luc (Upper) along with 250 ng of pcDNA 3.1 (EV), Runx2 (wild-type or Y428A mutant), and/or Smad3 or 5. A promoterless luciferase gene was used as internal control for the transfection efficiency. Cells were treated with TGF-β (10 ng/ml) or BMP2 (300 ng/ml) for 6 h and harvested in passive lysis buffer 24 h after transfection. Ten microliters of the cell lysate was used for dual luciferase assay. The activity of the firefly luciferase was normalized with that of Renilla luciferase. The graphs represent three independent experiments with n = 6. TβRE, TGF-β-responsive element.