Fig. 3. Discrete Ubx and Smad binding sites are closely juxtaposed, but independent of one another, in the sal1.1 CRE. Probes with Ubx binding sites 5 and 6 (red circles) and Med and Mad binding sites (green and yellow boxes, respectively) and mutated binding sites (marked with an `X') are depicted above each EMSA. Protein-DNA complexes are indicated by arrowheads. (A,B) EMSAs with either purified Ubx homeodomain (Ubx-HD) (A) or full-length Ubx1a (B) on wild-type and mutated sal probes. (A) Mutations in Ubx binding sites 5 and 6 cause a 10-fold decrease in Ubx-HD binding affinity for probe (compare lanes 1-7 with 9-14), but mutations in the kM1 site (lanes 16-21) have no effect on binding. (B) Mutations in Ubx binding sites 5 and 6 eliminate the binding of full-length Ubx protein to the probe (compare lanes 1-5 with 6-10), but mutations in the kM1 site (lanes 11-15) have no effect on binding. (C) Mutations in Ubx binding sites 5 and 6 do not affect Med binding affinity for its site, as compared with the wild-type probe (compare lanes 1-4 with 5-8). (D) Mutations in Ubx binding sites 5 and 6 do not affect Mad binding affinity for its site, as compared with the wild-type probe (compare lanes 9-13 with 14-18).