Figure 3

The mouse Tgif2d protein is a functional repressor. A) COS-1 cells were cotransfected with myc-tagged mSin3A and the indicated Flag-tagged expression constructs. Proteins were isolated on Flag agarose and analyzed for coprecipitating mSin3A with a myc antibody. Expression of transfected proteins in cell lysates is shown below. B) HepG2 cells were transfected with a reporter in which the luciferase gene is driven by the TK promoter and two TGIF binding sites, together with increasing amounts of mTgif2 or mTgif2d. Luciferase activity was assayed after 36 hours and is shown compared to a control transfection (mean +/- s.d. of triplicate transfections). C) COS-1 cells were cotransfected with a Smad2 or Smad3 expression vector and the indicated Flag-tagged expression constructs. Proteins were isolated on Flag agarose and analyzed for coprecipitating Smad2 or Smad3. Expression of transfected proteins in cell lysates is shown below. D) HepG2 cells were transfected with the 3TP-lux reporter, together with human TGIF, TGIF2 or mouse Tgif2d. After 24 hours, cells were treated with TGFβ (100 pM), or left untreated and luciferase activity was assayed 16 hours later, and is shown as the mean +/- s.d. of triplicate transfections.