GATA-1 specifically binds to 2 GATA motifs in subregion C. (A) Nucleotide sequence of subregion C. The sequence from position +5653 to +6154 is shown relative to the ATG translation start site of ABO. The sequence was derived from the genomic DNA of K562 cells (JN863720). The motifs for several relevant transcription factors and the E-box are indicated by over bars. The sequences typed in bold indicate the oligonucleotides G1 and G2 used in EMSA. The positions and identities of the mutations in the GATA motifs used in the EMSA and transfection experiments are represented by underlining and arrows. (B) Oligonucleotides derived from subregion C bind a nuclear protein. EMSAs were performed using the nuclear extracts from K562 cells. DNA-protein interaction was investigated using radiolabeled probes G1 and G2 in the presence or absence of a 100-fold molar excess of competing unlabeled oligonucleotides (lanes 1-4 and 6-9). Oligonucleotides GATA and mGATA represent a GATA consensus oligonucleotide and a mutant oligonucleotide containing “GA” to “CT” substitutions within the GATA motif, respectively. Mutated versions of oligonucleotides G1 and G2 and mG1 and mG2, respectively, were used as radiolabeled probes (lanes 5 and 10). The major shifted complex is indicated by an arrow. (C) Anti–GATA-1 Ab diminishes the DNA-protein complex. The nuclear extracts prepared from K562 cells were preincubated with anti–GATA-1 Ab (N6) or anti–GATA-2 Ab (CG2-96) before the addition of the radiolabeled probes.