Figure 8.

TGF-β/Smad3 signaling diminishes the binding of MyoD protein complexes to E-box DNA sequences. (A) Electrophoretic mobility shift assay (EMSA) using nuclear extracts from 10T1/2 cells transfected with Myc-tagged MyoD and Flag-tagged Smad3, as indicated, in the presence or absence of TGF-β. The oligonucleotide probe contains two direct repeats of an E-box sequence from the MCK enhancer (2xMEF1). In lane 4, anti-Myc antibody, which recognizes Myc-tagged MyoD, was added to the binding reaction. The MyoD-containing DNA-protein complex (Shift) was identified by comparing the gel-shift patterns from extracts of vector-transfected control and MyoD-transfected cells, and by the appearance of a supershifted (SS) tertiary complex in the presence of anti-Myc antibody. The composition of the lower mobility complexes, present in all samples, including control reactions, is not clear; however, these bands were absent when the oligonucleotide contained only one E-box sequence (data not shown). (B) Binding of MyoD to the MEF1 sites was analyzed using biotin-labeled oligonucleotides. The biotinylated wild-type 2xMEF1 (W) or a mutant MEF1 (M) oligonucleotide immobilized on streptavidin beads were incubated with lysates of the transfected 10T1/2 cells as in A, and the DNA-bound MyoD complexes were analyzed by gel electrophoresis followed by immunoblotting using anti-Myc antibody. The expression of MyoD or Smad3 in the cells was detected by immunoblotting of a fraction of the lysates. (C) EMSA was also performed for nuclear extracts of cells transfected with HA-tagged MyoD∼E47 fusion protein. Binding of this protein to the 2xMEF2 site, as identified by the supershifted band following incubation with anti-HA antibody, was not significantly affected by TGF-β treatment and Smad3 coexpression.