EGMSA using GST-+-MITF fusion protein or using the nuclear extract obtained from FMA/3 cells overexpressing +-MITF or mi-MITF. (A) Nonbinding of GST-+-MITF to the CAGTTG motif in the MMCP-5 promoter. The labeled 5′-ACAGAGGCACAGTTGAGGACCTAT oligonucleotide in the MMCP-5 promoter (oligo 1) or the labeled 5′-TGGTGGGGACACATGTTACATGGA oligonucleotide in the MMCP-6 promoter (oligo 3) was used as a probe (each hexameric motif is boxed in the figure). Competition for the binding of GST-+-MITF to the labeled oligo 3 was also examined. The excess amount of nonlabeled oligo 1 or oligo 3 was added. The binding was inhibited by nonlabeled oligo 3 but not by nonlabeled oligo 1. (B) EGMSA using nuclear extract obtained from FMA/3 cells overexpressing +-MITF or mi-MITF. Oligo 1 was used as a probe. The excess amount of nonlabeled oligo 1 or the nonlabeled oligonucleotide mutated at the CAGTTG motif (to CTGTAG, oligo 2) was added. The binding was inhibited by nonlabeled oligo 1 but not by nonlabeled oligo 2. (C) EGMSA using whole cell extract obtained from FMA/3 cells overexpressing +-MITF or mi-MITF. Oligo 1 was used as a probe. The excess amount of nonlabeled oligo 1 or oligo 2 was added.