Fig. 7. Mad and Drosophila CrebB proteins bind in vitro to the XC enhancer. (A) Gelshift experiments with Mad protein [50 ng (+) or 200 ng (++)] were performed on double-stranded oligonucleotides corresponding to wild-type or mutated (DRS2m) versions of DRS2, in the presence or absence of a 500-fold excess of cold DRS2 competitor. Lanes 1-3 show a dose-dependent binding of Mad to DRS2. Lanes 4-7 indicate that binding is competed by cold DRS2 (lanes 4 and 5), and that the integrity of the DRS2 site is required for Mad binding (lanes 6-7). (B) Similar experiments on double-stranded oligonucleotides corresponding to the wild-type or mutated (DRS1m) version of DRS1. Compared with the experiments in A, this gelshift shows that Mad protein binds DRS2 with a stronger affinity than DRS1. (C) Gelshift experiments with Drosophila CrebB protein [20 ng (+) or 100 ng (++)] were performed on double-stranded oligonucleotides corresponding to wild-type (Creb1-2) or mutated (Creb1-2m) versions of Creb-binding sites, in the presence or absence of a 500-fold excess of cold Creb1-2 competitor. Lanes 1-3 show a dose-dependent binding of Drosophila CrebB. Lanes 4-7 indicate that the binding of Drosophila CrebB is competed by the competitor, and that the integrity of the two Creb binding sites is required for binding to occur.