Fig. 5.

Binding of recombinant GST-ATAF2 fusion protein to the −117 to −82 fragment of NIT2 promoter in eletrophoretic mobility shift assay (EMSA). (A) For labeled probes preparation in EMSA, three tandemly arrayed 36 nucleotide sequences of NIT2 promoter (underlined, F1–F3) were selected to examine the specificity of binding. Lane 1, 500 ng of GST; lanes 2, 4, 6, 8, 50 ng of GST-ATAF2; lanes 3, 5, 7, 9, 150 ng of GST-ATAF2; lanes 2 and 3 contained ³²P-labeled F1 fragment as probe; lanes 4 and 5 contained the radio-labeled F1 fragment as probe and 100-fold unlabeled F1 fragment as competitor; lanes 6–7 and 8–9 contained ³²P-labeled F2 and F3 fragments as probes, respectively. (B) EMSA of the −117 to −82 fragment of NIT2 promoter incubated with the oligonucleotide carrying mutations as competitors. Shown are the sequences of the mutant oligonucleotides (M1–M9) corresponding to the −117 to −82 (F1) of NIT2 promoter which were used as competitors in EMSA. EMSAs were performed using ³²P-labeled F1 fragment as probe. Lane 1 contained cold-labeled F1 fragment as competitor; lanes 2–10 contained base-substituted mutant competitors shown in the box; lane 11, no competitor.