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Fig. 1. GFPSmad2, GFPSmad3 and GFPSmad4 fusion proteins form DNA-bound complexes that are transcriptionally active in response to TGF-ß. (A) HeLa TK^- cells expressing either GFPSmad3, GFPSmad4, HA-Smad4 or FLAG-Smad3 as indicated were either untreated or treated with TGF-ß1 for 1 hour. Cell extracts were assayed by bandshift analysis using the c-jun SBR as a probe. The positions of migration of complexes containing different combinations of tagged Smad3 and Smad4 are indicated. The same extracts were western blotted with antibodies recognising Smad3, phosphorylated Smad3 (P-Smad3) or Smad4 (below). The right-hand panel shows transcription assays in which MDA-MB468 cells were transfected with the CAGA₁₂-luciferase reporter gene, plasmids expressing the different GFPSmad fusion proteins and pEF-lacZ as an internal control for transfection efficiency. Cells were treated with or without TGF-ß1 for 8 hours. Luciferase was quantitated relative to ß-galactosidase from the pEF-lacZ internal control. The data are means and standard deviations of a representative experiment performed in triplicate. (B) HeLa TK^- cells were transfected with plasmids expressing XFoxH1a, GFPSmad2 or GFPSmad4 as indicated. Total cell extracts from cells either left untreated or treated with TGF-ß1 for 1 hour were assayed by bandshift analysis using the activin response element (ARE) as probe. DNA-bound complexes containing either XFoxH1a and endogenous Smad2 and Smad4 or XFoxH1a and GFPSmad2 and GFPSmad4 are indicated. The same extracts were western blotted with antibodies recognising Smad2/3, phosphorylated Smad2 (P-Smad2) or Smad4 (below). The right-hand panel shows transcription assays performed as above, where MDA-MB468 cells were transfected with the ARE₃-luciferase reporter gene, plasmids expressing XFoxH1a, the different GFPSmad fusion proteins and pEF-lacZ. The data are means and standard deviations of a representative experiment performed in triplicate.

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