FIG. 6.

Microphthalmia binds to TRP1 and TRP2 M and E boxes. (A) TRP1 M box (−44 to −33), TRP1 E box (−238 to −233), TRP2 M box (−129 to −135), and TRP2 E box (−346 to −340) were used as probes. Gel shift assays were performed in an in vitro transcription-translation reaction using empty pCDNA3 or pCDNA3 encoding microphthalmia (pCDNA3-MI). For competition experiments, unlabeled homologous and mutated oligonucleotides were added in 50-fold excess. (B) B16 nuclear extracts from control cells (−) or cells treated with forskolin for 6 h (+) were incubated with labeled TRP1 M box and TRP2 M box. Where indicated, reactions were carried out in the presence of preimmune serum (PI) or a specific antibody directed against the C terminus of the microphthalmia (α-MI). For competition experiments, unlabeled homologous and mutated oligonucleotides were added in 50-fold excess. Autoradiograms were exposed for 15 h at −80°C, except for competition experiments with TRP1 or TRP2 E box, which were exposed for 48 h.