AML1/Evi-1 physically interacts with Smad3 and inhibits the Smad3 activity. (A) Association between AML1/Evi-1 and Smad3 in vivo. Twenty micrograms of pME18S (lane 2), AML1/Evi-1 (lanes 1 and 3), AML1/Evi-1▵ZF1-7 (lane 4), or AML1/Evi-1▵Rep (lane 5) in pME18S was transfected into 4 × 106 of COS7 cells with 20 μg of the empty pCMV5 (lane 1) or Smad3-Flag in pCMV5 (lanes 2 to 5). Cells were obtained 48 hours later and subjected to the immunoprecipitation procedure with the anti-Flag antibody. Immunoprecipitates were resolved by SDS-PAGE, and detected by the anti–Evi-1 antiserum (top). Expression of Smad3-Flag and Evi-1 is monitored with the anti-Flag (middle) and the anti–Evi-1 (bottom) antibodies, respectively. The location of Smad3 is shown at right, and the positions of molecular-weight standard at left. (B) AML1/Evi-1 inhibits Smad3/4-induced TGF-β responses. Either the empty pME18S, Evi-1, AML1/Evi-1, or AML1/Evi-1▵ZF1-7 in pME18S in combination with p3TP-Lux was cotransfected into HepG2 cells together with pCMV5 or Smad3 plus Smad4 (Smad3/4). Relative luciferase activities were measured in cell extracts, normalized to the β-galactosidase activity. Values and error bars depict the means and the standard deviations, respectively, of four separate experiments.
