TGF-β Signaling Cooperates with AT Motif-Binding Factor-1 for Repression of the α-Fetoprotein Promoter
Figure 7
SBE in the proximal region of the AFP promoter is not essential for TGF-β-mediated suppression of the AFP promoter. (a) Schematic presentation of 3′52mSBELuc. A putative SBE, 5′-CAGATA-3′, was mutated to 5′-TTTTTT-3′. (b) Effect of TGF-β on 3′52Luc and 3′52mSBELuc activities. HepG2 cells were transfected with each reporter plasmid (200 ng). The cells were then stimulated with 5 ng/mL TGF-β. (c) Indirect binding of Smad3 to an AT-motif via ATBF1. The indicated cell lysates were mixed with biotinylated AT-motifs. Then, streptavidin-agarose was added to the mixture. Subsequently, a protein(s) bound to streptavidin-agarose was purified. After SDS-PAGE, IB was carried out using anti-Flag or anti-HA antibody. Using the total lysates, each protein was detected with anti-Flag, anti-HA, anti-V5, or anti-phosphorylated Smad3 (PS3) antibody.
