Figure 2.

FOXO3a directly binds to the TR4 promoter. A, [³²P]-labeled TR4-DBE probe and in vitro-translated FOXO3a protein were used in EMSA. FOXO3a-specific antibody, the double-stranded DNA oligonucleotide of TR4-DBE, or its single point-mutant TR4-DBEm were added in EMSA (lanes 4, 2, and 3, respectively). B, DNA pull-down assays used [³⁵S]-labeled in vitro-translated FOXO3a protein with biotinylated, double-stranded DNA oligonucleotides encompassing FOXO DBE of the MnSOD promoter (SOD-DBE), FOXO DBE of the TR4 promoter (TR4-DBE), and its point mutant (TR4-DBEm) in lanes 2, 3, and 4, respectively. Lane 1, input control protein alone. C, ChIP assay showed FOXO3a bound to the TR4 promoter in H1299 cells. The DNA protein complexes were cross-linked with formaldehyde and subjected to ChIP assay using an anti-FOXO3a antibody or rabbit IgG (negative control) and the primers to amplified DBE and control primers. The primer sequences and location were indicated in Materials and Methods.