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Fig. 4. Zen binding is enhanced in the presence of Mad. (A) Schematic representation of the 42 bp wild-type (wt-42) and mutant oligonucleotides that eliminate the core Smad-(Sm-42) or Zen-(Zm-42) binding sites, and the 128 bp DNA fragment from the Race enhancer used in electrophoretic mobility shift assays (EMSAs) showing Medea-(blue circle), Mad-(blue ovals) and Zen-(red ovals) binding sites. (B) DNA binding requires the core consensus sites. ³²P-labeled wild type (lanes 1-4) and mutant oligonucleotides (Sm-42, lanes 5-8; Zm-42, lanes 9-12) were incubated with no protein (lanes 1,5,9); and 100 ng Mad (lanes 2,6,10), 100 ng Medea (lanes 3,7,11) or 10 ng Zen (lanes 4,8,12). Mad and Medea produce a single complex of bound probe, whereas Zen produces two complexes. The slower migrating Zen complex could be due to a Zen/Zen/DNA complex. Mutant Sm-42 (or Zm-42) eliminated the binding of Mad/Medea (or Zen) without affecting the binding of Zen (or Mad/Medea). (C) ³²P-labeled wild-type DNA fragments were incubated with no protein (lanes 1 and 5), increasing amounts of Mad (1 ng, 2 ng, and 5 ng in lanes 2-4, respectively), increasing amounts of Zen (0.1 ng, 0.3 ng, and 1 ng in lanes 6-9, respectively) or increasing amounts of Zen in the presence of 1 ng of Mad (lanes 10-13). The amounts of Zen in lanes 10-13 were the same as in lanes 6-9. More Zen complexes are shifted in lanes 10-13 compared with lanes 6-9. In lane 13, a supershifted complex (arrow) is visible above the Zen complex (arrowhead) that may be due to the formation of a Mad/Zen/DNA complex or a Zen/Zen/DNA complex. (D) Similar experiment as in C, except that Zen-Del was used instead of wild-type Zen. The same amounts of proteins were used as in C. Enhancement of Zen binding by Mad was not observed.

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