FIG. 5.

Direct binding of Smad3 and Smad4 to the MMTV LTR sequence −160/−143 containing an AGACA (Smad) box. (A) EMSA with E. coli-expressed GST-Smads or GST control alone (lane 1) incubated with ³²P-labeled probes encompassing the putative Smad box (without fp1/fp2 [Smad B.S. wt]) or a mutated one (mt, lane 7; see Fig. 1A). ns, nonspecific. (B) EMSA with E.coli-expressed GST-Smads and the wild-type Smad box probe (wt, lanes 1 to 3 and 5 to 7) or the mutated box (m, lanes 4 and 8). Unlabeled, double-stranded oligonucleotides were added in ∼300-fold excess over the probe (lanes 2 and 6). Their sequences were as follows: wild type (wt) or an unrelated (u) hepatic nuclear factor 3 binding site. (C) DNA precipitation assay. Lysates of COS cells expressing Flag-Smad3 with (lanes 2 to 4) or without (lane 1) an activated TGF-β receptor (T204D) were incubated with biotinylated, double-stranded Smad-binding site probes, either wild type (wt, lanes 1 to 3) or mutated (mut, lane 4), and precipitated with streptavidin magnetic beads. DNA-bound precipitates (DP; upper panels) or aliquots of input lysates (lower panels) were subjected to immunoblot analysis with an anti-Flag antibody. Lanes 1 and 2 and lanes 3 and 4 are from two independent experiments.