Cytoplasmic retention of NF-P motif–binding factors in CTLL-2 cells overexpressing mi-MITF.

(A) Two nuclear factors that specifically bind the NF-P motif are present in the nucleus of CTLL-2 cells. The nuclear extract (NE) was prepared from CTLL-2 cells and incubated with ³²P-labeled oligonucleotide NF in the presence or absence (−) of the competitor. For competition, NE was incubated with an excess amount (100-fold) of unlabeled oligonucleotide NF or oligonucleotide mNF prior to the reaction with the probe. The reaction mixture was separated by polyacrylamide gel. Arrowheads 1 and 2 indicate the same retarded bands that specifically bind the NF-P motif. (B) The nuclear (NE) and cytoplasmic (CE) fractions of CTLL-2 cells were examined with EGMSA by using oligonucleotide NF as a probe. Arrowheads 1 and 2 indicate the same retarded bands that specifically bind the NF-P motif. (C) EGMSA of CTLL-2 cells transfected with +-MITF and mi-MITF. The nuclear and cytoplasmic fractions were extracted from CTLL-2 cells that had been transfected with +-MITF, mi-MITF, or vector alone (Vec) 5 days before. The extracts were incubated with³²P-labeled oligonucleotide NF, and separated on polyacrylamide gel. Arrowheads 1 and 2 indicate the same retarded bands that bind specifically the NF-P motif. (D) Gene expression in CTLL-2 cells transfected with cDNA encoding +-MITF or mi-MITF. CTLL-2 cells were transfected with +-MITF, mi-MITF, or vector alone. After 5 days, total RNA (3.0 μg) was extracted from the cells, blotted, and hybridized with the MITF, Gr B, and perforin probes. Reprobing with β-actin probe verified an RNA equal loading.