Fig. 3. CES-1 binds to the Snail-binding sites/E-boxes in Region B of the egl-1 locus in vitro. A GST-CES-1 fusion protein binds to wild-type but not mutant Region B in vitro. Increasing amounts of bacterially expressed, affinity-purified GST-CES-1 fusion protein [0 mol (lanes 1, 7 and 13), 8x10^-13 mol (lanes 2, 8 and 14), 2x10^-12 mol (lanes 3, 9 and 15), 4x10^-12 mol (lanes 4, 10 and 16), 6x10^-12 mol (lanes 5, 11 and 17), 8x10^-12 mol (lanes 6, 12 and 18) were incubated with 7 ng of radioactively labeled wild-type Region B with four intact Snail-binding sites (lanes 1-6), or mutant Region B with all four Snail-binding sites mutated to Snail^-/E-box^- sites (lanes 7-12) or to Snail^-/E-box⁺ sites (lanes 13-18). Electrophoretic mobility shift assays were performed as described in the Materials and Methods. Asterisks indicate protein-DNA complexes with one, two or three CES-1 molecules bound to Region B. Triangle indicates a protein-DNA complex that is most likely a bacterial contaminant bound to Region B.