FoxK binds to specific DNA sequences and regulates transcription. (A) EMSA performed with GST-FoxK(414–654) or GST alone (protein [P]) and the radiolabeled double-stranded oligonucleotide Oligo-FH containing an optimum FH-binding site (red). Cold Oligo-FH probe (FH), a suboptimum FH-binding site (Sub), and an unrelated sequence (GAS) were used at 100-fold molar excess (competitors [C]). The higher bands (arrows) indicate specific binding of FoxK to Oligo-FH. Cold Oligo-FH efficiently competes for FoxK, whereas a suboptimum FH-binding site is a less efficient competitor and GAS does not compete for FoxK. GST alone did not bind to Oligo-FH. Free oligonucleotides complexes accumulate in the bottom (arrowhead). (B and C) A plasmid driving luciferase under the control of six consecutive Oligo-FH sequences (6xFH) was cotransfected with pAc5C-FoxK-L-V5, pAc5C-FoxK-S-V5, or empty vector in S2 cells. As expected, both FoxK-L and FoxK-S isoforms exhibit nuclear localization in transfected S2 cells (green, FoxK-L-V5). (D) Both FoxK-L and FoxK-S induce a fourfold activation of the 6xFH target sequence. The error bars correspond to the standard deviation of three independent experiments. (E) The FoxK-L and FoxK-S proteins migrate in two distinct bands in Western blot, suggesting posttranslational modification. β-Galactosidase was used for normalization.