Fig. 6. Esr10 proneural enhancer activity requires intact E-boxes. HeLa cells were transfected with expression vectors for ICD, Xngnr1 and E47 (N/E), or both, and with luciferase vectors driven by wild-type (Esr10/Dra) or mutated (Esr10/DramE1E2) elements, shown schematically above (A). In the case of Esr10/Dra, ICD and N/E synergistically activate transcription approximately three times more over ICD alone (A, left). Synergy was lost when E-boxes were mutant (A, right). E-box motifs were also required for GFP expression driven by Esr10/Dra in transgenic frogs (compare C with B). (D,E) Injection of Xngnr1 mRNA with a lacZ tracer into Esr10/DramE1E2 transgenic embryos (E) could not activate enhancer activity as was seen with controls.(F) EMSA showing that Xngnr1 (N) and E47 (E) proteins shift an E2 oligo; shifts were competed by 10x and 100x cold competitor (WT) but not by similar increases mutant E2 oligos (Mut) or oligos corresponding to a binding site of a heterologous activator (Vax) (Mui et al., 2005). O, oligo; R, reticulocyte lysate; N/E, Xngnr1 plus E47. Complexes formed by E47 homodimers (Ex2) are of higher mobility than those formed by Xngnr1/E47 (N/E) heterodimers. ns, nonspecific complexes attributable to reticulocyte proteins.