EGMSA using oligonucleotides containing E-box or NF-P motif.
(A) Sequences of oligonucleotides used in EGMSA. P6 contains the CACATG motif (boxed) of the MMCP-6 gene promoter, which is mutated (underlined) in mP6. E1 and E2 contain the CAACTG and CAGCTG motifs (boxed) of the perforin gene promoter, respectively. NF contains the NF-P motif (boxed) of the perforin gene promoter, which is mutated (underlined) in mNF. Arrowheads indicate the position of nucleotides in the promoters. (B) In vitro binding of MITF to the 2 CANNTG motifs (E1 and E2) and the NF-P motif in the perforin promoter. GST-fused +-MITF or mi-MITF protein was incubated with32P-labeled oligonucleotides (probe oligonucleotide) in the presence or absence (−) of the competitor. For competition, an excess amount (100-fold) of indicated unlabeled oligonucleotides was added to the reaction prior to the probe. The reaction mixture was separated on polyacrylamide gel. An arrowhead indicates the position of protein-DNA complex.
