FIG. 8.

Smad7 interferes with the DNA-binding activities of Smad2 and Smad4. (A) Smad7 decreases the DNA-binding activities of Smad2 and Smad4. Flag-Smad4, Flag-Smad2, and Myc-FoxH1 were cotransfected with Smad7 into HEK293T cells. At 48 h posttransfection, the cells were treated with 100 pM TGF-β1 for 2 h. Then, cell lysates were harvested for an oligonucleotide precipitation assay with the biotin-labeled ARE oligonucleotide. DNA-bound proteins (top) and total protein levels (middle and bottom) were analyzed by immunoblotting using anti-Flag or anti-Myc antibodies. (B) Smad7 attenuates TGF-β-induced formation of the Smad-DNA complex containing Smad2, Smad4, and FoxH1. HEK293T cells were transfected with expression plasmids as indicated. At 48 h posttransfection, the cells were treated with 100 pM TGF-β1 for 2 h. EMSA was performed with nuclear extracts (top). Protein expression was analyzed by immunoblotting (Western blotting [WB], middle and bottom). (C) Recombinant GST-Smad7 MH2 protein reduced TGF-β-induced formation of the functional Smad-DNA complex. HEK293T cells were transfected with expression plasmids as indicated and harvested after 48 h. EMSA was performed with whole-cell extracts. GST-Smad7 MH2 or GST protein (0.5 μg) was used.