Fig. 5. HLH-2/HLH-3 binds to the Snail-binding sites/E-boxes in Region B of the egl-1 locus in vitro. (A) A HLH-2 homodimer and HLH-2/HLH-3 heterodimer bind to wild-type Region B in vitro. Increasing amounts of bacterially expressed, affinity-purified His₆-tagged HLH-2 (lanes 2-6), HLH-3 (lanes 7-11) or both HLH-2 and HLH-3 (lanes 12-16) fusion proteins [0 mol (lane 1), 8x10^-14 mol (lanes 2, 7 and 12), 2x10^-13 mol (lanes 3, 8 and 13), 4x10^-13 mol (lanes 4, 9 and 14), 8x10^-13 mol (lanes 5, 10 and 15), 2x10^-12 mol (lanes 6, 11 and 16)] were incubated with 7 ng of radioactively labeled wild-type Region B. Electrophoretic mobility shift assays were performed as described in the Materials and Methods. Asterisks indicate a DNA-protein complex with one or two heterodimers bound to Region B. (B) A HLH-2/HLH-3 heterodimer still binds to Snail^-/E-box⁺ binding sites in Region B. Increasing amounts of both His₆-tagged HLH-2 and HLH-3 [0 mol (lane 1, 7 and 13), 8x10^-14 mol (lanes 2, 8 and 14), 2x10^-13 mol (lanes 3, 9 and 15), 4x10^-13 mol (lanes 4, 10 and 16), 8x10^-13 mol (lanes 5, 11 and 17), 2x10^-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 Materials and Methods. Asterisks indicate a DNA-protein complex with one or two heterodimers bound to Region B.