Figure 5.

Binding activity of MITF extracted from human mast cells to CACCTG motifs in the 5 flanking region of the tryptase gene. The nuclear extracts purified from HMC-1 cells were incubated with biotinylated Oligo-1 (WT-1) or -3 (WT-2) containing the potential MITF-binding site as probe for EGMSA. After electroporesis, DNA-protein complex was transferred to nylon membrane and blotted with avidin-HRP for visualization. CACCTG motifs of WT-1 and -2 were modified as shown in Mut-1 and -2 for competitive DNA binding assay, respectively, and anti-MITF mAb was added for supershift, as described in the Methods.