HER-2 overexpression does not alter transforming growth factor (TGF)-β1 induced Smad2 activation or nuclear accumulation or the formation of Smad-Smad-binding element (SBE) complexes. (a) TGF-β1 binds equally well to the surface of MCF-7 CN and MCF-7 H2 cells. The binding of fluorescein isothiocyanate (FITC)-labeled recombinant TGF-β1 was monitored by flow cytometry. The red curves show the FL1 values for the untreated control cells, the purple (filled) curves indicate the intensity of cells incubated with an irrelevant FITC-labeled protein and the green curves show the intensity of cells treated with FITC-labeled recombinant TGF-β1. The median shift in FL1 values (fold increase) between the irrelevant control protein and TGF-β1 is indicated. (b) TGF-β1 induced phosphorylation of Smad2 is detected in both MCF-7 CN and MCF-7 H2 cells. A phospho-specific Smad2 antibody was used on immunoblots of whole cell lysates from diluent or TGF-β1 treated cells. The blot was re-probed with an antibody against total Smad2, which cross-reacts weakly with Smad3. (c) Endogenous Smad2 is concentrated in the nucleus after exposure to TGF-β1 in MCF-7 or MDA-MB-231 cells with or without HER-2 overexpression. Vector control (CN) cells (left panels) or HER-2 overexpressing (H2) cells (right panels) were treated for 1 h with either diluent control or 2 ng/ml TGF-β1, fixed and stained with the anti-Smad2 antibody. The endogenous Smad2 protein was visualized with either a peroxidase/3,3'-Diaminobenzidine (DAB) reaction (top two rows) or an Alexa-488 conjugated secondary antibody (bottom row). (d) TGF-β1 stimulated Smad DNA binding is not affected by HER-2 overexpression in MCF-7 cells. Nuclear protein extracts from MCF-7 parental (PAR), MCF-7 CN and MCF-7 H2 cells treated with either diluent or TGF-β1 were reacted with biotin labeled oligonucleotides containing SBEs. Avidin-coupled sepharose beads were used to collect the DNA-protein complexes. The Smad composition of the complexes was analyzed by western blotting with anti-Smad antibodies as indicated. The last two lanes (CNmt) contained the same MCF-7 CN nuclear extract as in lanes 3–4, with a mutant oligonucleotide in which one of the SBE sites was mutated (i.e. PE2Sm1 ).