Figure 5.

(A and B) Endogenous or transfected Smad3 cannot bind to the SBE in the absence of Smad4, and the Smad3–Smad4 complex binds optimally to the SBE. (A) SW480.7 cells were cotransfected with Flag-Smad2, Flag-Smad3, and Smad4-HA, and treated with TGF-β as indicated. Whole-cell extracts were used in a gel mobility shift assay with the 16-bp probe. (B) Antibodies against the Flag and HA epitopes were included for supershift assay. (C) Recombinant Smad3 and Smad4 can bind to the 16-bp probe. GST-SMAD proteins (0.8 μg) was used in a gel mobility shift assay.