Fig. 7. The Gata4 lateral mesoderm enhancer contains two high-affinity GATA-binding sites. (A,B) Recombinant GATA4, GATA5 and GATA6 proteins were transcribed and translated in vitro, and used in EMSA with radiolabeled double-stranded oligonucleotides encompassing the Gata4 gata I site (A, lanes 2-7) or the gata II site (B, lanes 2-7). In A and B, lane 1 contains reticulocyte lysate without recombinant protein (represented by a minus sign). GATA4 efficiently bound to both the gata I and gata II sites (A and B, lane 2) and this binding was specifically competed by excess unlabeled gata I (A, lane 3) and gata II probes (B, lane 3). GATA5 and GATA6 proteins also bound, although more weakly than GATA4 protein, to the gata I (A, lanes 4,6) and gata II sites (B, lanes 4,6), and binding by GATA5 and GATA6 was specifically competed by excess unlabeled gata I (A, lanes 5,7) and gata II probes (B, lanes 5,7). Approximately equivalent amounts of GATA4, GATA5 and GATA6 proteins were used in each sample. (C) Recombinant GATA4 protein was transcribed and translated in vitro and used in EMSA with a radiolabeled double-stranded oligonucleotide encompassing the Gata4 gata I site (lanes 1-5) or the gata II site (lanes 6-10). Lanes 1 and 6 contain reticulocyte lysate without recombinant GATA4 protein (represented by a minus sign). GATA4 efficiently bound to both the gata I and gata II sites (lanes 2,7). Binding of GATA4 to the Gata4 gata I site was competed by an excess of unlabeled gata I site (I, lane 4) and by an excess of an unlabeled control GATA site from the Nkx2.5 gene (C, lane 3), but not by an excess of a mutant version of the Gata4 gata I site (mI, lane 5). Likewise, the binding of GATA4 to the Gata4 gata II site was specifically competed by excess unlabeled gata II probe (II, lane 9) and by excess unlabeled control probe (C, lane 8), but not by an excess of a mutant version of the gata II probe (mII, lane 10).