Activation of the Cox2 promoter by BMP signaling. (A) Depiction of 4 luciferase reporters of the Cox2 promoter, −8653 to +53 bp, −7444 to +53bp, −1500 to +1 bp, and −371 to +70 bp. Putative Smad-binding sites are denoted. (B) MECs were transfected with luciferase reporter plasmids carrying different lengths of the Cox2 promoter in the presence or absence of BMP6 treatment or the cotransfection of an active Smad1 expression vector. After 48 hours, cells were lysed to measure luciferase activity. Luciferase activity was normalized using Renilla luciferase activity. Data shown are the means ± SD of triplicates. (C) An end-labeled fragment containing the 29-bp Smad-responsive region was incubated with nuclear extracts from MECs and with different nonlabeled competitors. Complexes were resolved on a nondenaturing 4% polyacrylamide gel followed by autoradiography of the dried gel. The addition of nuclear extract produced a specific shifting band denoted as I. *Nonspecific band. S indicates specific; NS, nonspecific; and SBE, Smad-binding element. (D) Nuclear extracts were extracted from MECs treated with BSA or BMP6 or cotransfected with Flag-Smad1 and Flag-Smad4. Overexpression of Smads induced a slower-migrating complex labeled as II.