DNA binding ability of +-MITF, mi-MITF,or-MITF, or wh-MITF examined by EGMSA and dissociation assay performed with +-MITF or wh-MITF. (A) EGMSA using GST-MITF fusion proteins. The labeled 5′-TGGTGGGGACACATGTTACATGGA oligonucleotide was used as a probe (hexameric motif recognized by MITF is underlined). Each lane contains 3.5 μg of GST-+-MITF, GST-mi-MITF, GST-or-MITF, or GST-wh-MITF in the absence or presence of a 200-fold molar excess of unlabeled oligonucleotide as a competitor. Coomasie brilliant blue staining for GST-+-MITF, GST-mi-MITF, GST-or-MITF, or GST-wh-MITF was shown below the result of EGMSA. The amount of protein in each lane was the same as the amount used in EGMSA. (B) Dissociation assay performed with +-MITF and wh-MITF. Reaction mixtures for DNA-+-MITF and DNA-wh-MITF complexes were prepared, and a 500-fold molar excess of unlabeled oligonucleotide was added. The reactions were sampled after further incubation time of 30, 60, 90, 120, and 150 seconds. The regions of the autoradiogram containing DNA-MITF complexes were scanned with a densitometer, and the values were plotted against the incubation period.