BMP/SMAD1 represses the NODAL pathway through competition for SMAD4. (A) The AC, composed of transfected SMAD2, CA-ALK5, and FOXHI vectors, activates the (n2)7-luciferase reporter 30-fold in 293T cells. Activation of the BMP pathway with transfected SMAD1 and CA-ALK6 shows no response. (B,C) CA-ALK6 vector represses AC-dependent activation of (n2)7-luciferase (B), which is rescued by Smad4 vector in a dose-dependent manner (C). Ten to 200/300 indicate amount of transfected DNA in nanograms. (D) EMSA analysis of DNA complexes bound to the n2 FOXH1-binding site oligonucleotide with and without activation by CA-ALK5 or CA-ALK6 (see the text). Proteins encoded by transfected vectors, competitor oligonucleotides, and antibodies used for supershift analysis are indicated above. Asterisks represent MYC–FOXH1/n2 DNA/anti-MYC antibody supershifted complexes. ARE indicates the complex composed of pSMAD2/3, FOXH1, and n2 DNA. (ARE) Activin responsive element; (n2) specific competitor; (NS) nonspecific competitor; (NSB) nonspecific bands.