Fig. 5. Two evolutionarily conserved GATA sites in the Mef2c anterior heart field enhancer are bound by GATA4. GATA4 was transcribed and translated in vitro and incubated with radiolabeled, double-stranded oligonucleotides spanning the Mef2c GATA-p site (lanes 9-14), the Mef2c GATA-d site (lanes 15-20) or the gs1 GATA site from the Nkx2.5 gene as a control (lanes 1-8). GATA4 efficiently bound to all three GATA sites (lanes 2, 10 and 16). Binding of GATA4 to the GATA-p site was specifically competed by excess unlabeled Mef2c GATA-p (Gp) (lane 13) and by excess unlabeled Nkx2.5 gs1 (lane 11) but not by a 100-fold excess of a mutant GATA-p site (Mp) or by a 100-fold excess of a mutant Nkx2.5 gs1 site (M1) (lanes 12 and 14). Likewise, the binding of GATA4 to the GATA-d site was specifically competed by excess unlabeled GATA-d (Gd) (lane 19) and by excess unlabeled Nkx2.5 gs1 (lane 17) but not by a 100-fold excess of either mutant GATA-d (Md) or mutant gs1 (M1) (lanes 18 and 20). The Mef2c GATA-p (Gp) and GATA-d (Gd) sites also efficiently competed for GATA4 binding to the control gs1 GATA site from the Nkx2.5 enhancer (lanes 5 and 7), but mutant versions of the GATA-p (Mp) and GATA-d (Md) sites were unable to compete for binding to the Nkx2.5 gs1 site (lanes 6 and 8). In samples where in vitro translated GATA4 protein was not included (denoted by a minus sign in lanes 1, 9 and 15), an equal amount of unprogrammed reticulocyte lysate was included (lysate-derived bands are noted).