Fig. 1
From: FOXM1 binds directly to non-consensus sequences in the human genome
Mutation of the FOXM1 DBD inhibits DNA binding. a Sequence alignments of the DBD for a number of Forkhead family members with the secondary structure shown schematically above. The residues used to generate point mutations are outlined in red. (*) conserved amino acids. H1-3 are α-helices, the orange arrows are β strands, and W1-2 are winged domains. b Plot showing relative change of polarization of a fluorescently-labeled (6FAM) dsDNA FKH consensus oligonucleotide upon addition of increasing concentrations of GST-FOXM1 WT or mutant DBD proteins. The FP assay provides a quantitative method and non-disruptive method to determine FOXM1 affinity for target by measuring the fluorescence polarization signals from the FAM-labeled FKH consensus (see Materials and methods). Data are plotted as % binding and show mean ± SD of triplicate experiments. (WT Kd = 1.10 ± 0.02 μM, H287A Kd = 3.04 ± 0.10 μM). c Plots showing relative luciferase activity of a 6X DB-TATA-luciferase reporter in cells transiently transfected with either WT or DBD mutant FOXM1 with the T596A mutation as a positive control. Data are shown as fold induction of luciferase activity following doxycycline induction. d Plot showing fold induction of a luciferase reporter containing a 200 bp sequence taken from the CCNB1 promoter following doxycycline induction of WT and mutant FOXM1 expression. Data represent triplicate experiments ± SD. (*) P <0.05, (**) P <0.01, (***) P <0.001