Luciferase assay and EGMSA.

(A) Transactivation of the SgIGSF promoter by +-MITF. The promoter sequences of the SgIGSF from −1501 to +19 were inserted upstream of the luciferase (LUC) gene of the pSPLuc vector. In one construct, the CATTTG motif in the promoter was mutated to CTTTAG (indicated by X). The deletion construct lacking the CATTTG motif was also made. These reporter constructs were cotransfected into MST (upper panel) or Jurkat (lower panel) cells with the empty pEF-BOS vector or the vector containing either +-MITF ormi-MITF cDNA. Mean values of the luciferase activity obtained with triplicate samples were plotted; bars indicate SE. In most cases, the SE values were too small to be shown by bars. (B) In vitro binding between the CATTTG motif and +-MITF. Oligonucleotide E was labeled with α-³²P dCTP and used as a probe. The unlabeled oligonucleotides E and mE were used as competitors. The probe was incubated with either GST or GST–+-MITF protein in the absence (−) or presence of an excess amount of either competitor. An arrow indicates a protein-DNA complex.