Fig. 2. XFast-3 is the transcription factor component of endogenous ARF2. (A) Whole cell extracts were prepared from NIH3T3 cells transfected with either Flag-tagged XFast-1 or XFast-3 that were either treated or not with TGFß1 as indicated. Extracts were analysed by bandshift assay on the ARE and complexes supershifted with monoclonal antibodies against Flag, Smad2/3 or Smad4. The Smad-containing complexes ARF1 and ARF2 are indicated, as are the supershifted complexes and the complex of XFast-1 with DNA. (B) NIH3T3 cells were transfected with ARE-luciferase, EF-lacZ as an internal control and plasmids expressing transcription factor (XFast-1 or XFast-3), as indicated. Cells were treated with TGFß1 for 8 hours, then harvested and luciferase activity was measured relative to ß-galactosidase activity from the internal control. The data are averaged from six independent experiments and standard deviations are shown. (C) Nuclear extracts were prepared from uninjected Xenopus embryos or embryos overexpressing activin at the times indicated post stage 8 and analysed by bandshift assay on the ARE. Antibodies against Smad2 (S2), XFast-1 (F1) or XFast-3 (F3) were included in the bandshift reaction where indicated, alone or with 5 µg peptide to which the antibody had been raised. ARF1 and ARF2 are indicated, as are supershifted complexes. Arrowhead indicates complex resulting from the XFast-1/3 antisera alone binding the probe; competing peptides have no effect on this complex.
