Fig. 2
Biochemical characterization of the interaction between CBX and the FOXO3-DBD protein. a The dissociation constant (Kd) for the protein/ligand pair FOXO3-DBD/IRE-FAM was determined by FPA using the IRE-FAM oligonucleotide (5 nM) and increasing concentrations of the FOXO3-DBD protein (1–350 nM). b The IC50-value of CBX was determined by FPA in a competitive binding experiment with constant concentrations of the FOXO3-DBD protein (25 nM) and the IRE-FAM oligonucleotide (5 nM). The IC50-value was calculated by nonlinear least-square analysis. The binding affinity (Ki) value of CBX was calculated by the equation of Nikolovska–Coleska. c Binding of CBX to the FOXO3-DBD protein was analyzed by FAM-EMSA. 1 µM recombinant FOXO3 and 100 nM fluorescence-labeled FoxP3- or FoxP3-mutated oligonucleotides were incubated with increasing concentrations of CBX for 30 min at room temperature. In the sample marked with (-) no FOXO3-DBD protein was added. Densitometric analysis of the FOXO3-DBD/FoxP3 complex signal was done with the ImageJ 1.48 software. The untreated control was set as 100%. d FAM-EMSA was performed using 30 µg whole-cell extracts of SH-EP/FOXO3 cells treated with 50 nM 4OHT to activate FOXO3(A3)ERtm in combination with indicated concentrations of CBX for four hours. The whole-cell extracts were incubated with 100 nM fluorescence-labeled FoxP3- or FoxP3-mutated oligonucleotides for 30 min at room temperature. As indicated, cells were cultivated under standard (10% FCS) or serum starved (0.1% FCS) conditions. Equal loading of cellular protein extracts was ensured by immunoblot analysis with the anti-GAPDH antibody. Densitometric analysis of the FOXO3-DBD/oligonucleotide complex signal was done with the ImageJ 1.48 software. The controls without CBX treatment were set as 100%
