Fig. 6. The Gata4 lateral mesoderm enhancer contains a high-affinity Forkhead-binding site. (A) Recombinant FOXF1 protein was transcribed and translated in vitro and used in EMSA with a radiolabeled double-stranded oligonucleotide encompassing the Gata4 Fox I site (lanes 2-5). Lane 1 contains reticulocyte lysate without recombinant FOXF1 protein (represented by a minus sign). FOXF1 efficiently bound to the Gata4 Fox I site (lane 2) and this binding was efficiently competed by a range of excess unlabeled Gata4 Fox I oligonucleotides (lane 3-5). A nonspecific lysate-derived band is denoted. (B) Recombinant FOXF1 protein was transcribed and translated in vitro, and used in EMSA with a radiolabeled double-stranded oligonucleotide representing a canonical FOXF1 site (Control FoxFI site). Lane 1 contains reticulocyte lysate without recombinant FOXF1 protein (represented by a minus sign). FOXF1 efficiently bound to the control Fox I site (lane 2). This binding was efficiently competed by an excess of unlabeled control probe (lane 3) and by an excess of Gata4 Fox I probe (lanes 4-6), even when only a 10-fold excess of the competitor was present (lane 6). A mutant version of the Gata4 Fox I site failed to compete for FOXF1 binding even at a 100-fold excess (lane 7). (C) Recombinant FOXA2 protein was transcribed and translated in vitro, and used in EMSA with a radiolabeled double-stranded oligonucleotide representing a canonical control FOXA2 site (lanes 2-5) or the Gata4 Fox I site (lanes 7-10). Lanes 1 and 6 contain reticulocyte lysate without recombinant FOXA2 protein (represented by a minus sign). FOXA2 efficiently bound to the control FOXA2 site (lane 2) and to the Gata4 Fox I site (lane 7), and this binding was efficiently competed by an excess of unlabeled control FOXA2 probe (lanes 3 and 8) and by an excess of Gata4 Fox I probe (lanes 4 and 9), but not by an excess of unlabeled mutant Gata4 Fox I probe (lanes 5 and 10).