EGMSA using GST-+-MITF fusion protein. (A) Six kinds of oligonucleotides (oligo 1 to 6) were synthesized: oligo 1 to 5 were derived from the Gr B gene promoter and oligo 6 from the gp49B gene promoter.50 In vitro binding of oligo 6 to +-MITF was confirmed in the other experiment (data not shown). CANNTG motifs are boxed and the mutations introduced into the motifs are shown by underlines. The labeled oligonucleotide containing the CAGATG motif (oligo 1), the CACGTG motif (oligo 3), or the CATTTG motif (oligo 4) was used as a probe. The probes were incubated with purified GST-+-MITF fusion protein in the presence or absence of unlabeled oligonucleotide competitors. To compete the in vitro binding between the three CANNTG motifs (oligo 1, 3, and 4) and GST-MITF fusion protein, excess amount of unlabeled oligo 2, oligo 5, or oligo 6 was added. The arrowhead indicates the complex obtained with the labeled oligonucleotides and GST-MITF fusion protein. (B) Five kinds of oligonucleotides (oligo 7 to 11) were synthesized based on the promoter sequence of the TPH gene. The labeled oligonucleotide containing the CAAGTG motif (oligo 7), the CAGATG motif (oligo 8), the CAACTG motif (oligo 9), or the CAGGTG motif (oligo 10) was used as a probe. Competition for the binding of GST-+-MITF to the labeled oligo 10 was also examined. The excess amount of a nonlabeled oligo 6 or an oligonucleotide mutated at the CAGGTG motif (to CTGGAG, oligo 11) was added.