Fig. 4. The Mesp2/E47 heterodimer binds to the E3 site of the Epha4 enhancer. (A) The results of EMSA using nuclear extracts from NIH3T3 cells transfected with FLAG-Mesp2 and/or Myc-E47 and incubated with E3 probe. The quantities of nuclear extracts used (µg) are indicated. (B) A competition assay indicating the specificity of the binding of the Mesp2/E47 (2 µg each) complex to the E3 probe. The addition of 100-fold excess of unlabeled E3 probe, but not the E3m mutant probe, abolished the binding. (C) Evidence for the heterodimer formation of FLAG-Mesp2/Myc-E47. The band containing E3 (arrow) was supershifted by the addition of either anti-FLAG or anti-Myc antibodies. The oligonucleotides used were as follows: E3, 5'-TGGGTCACATTTGTCCAAAA-3'; E3m, 5'-TGGGTCAACGGGTTCCAAAA-3' (E-box is shown in the bold; altered nucleotides in the mutant probe are underlined).