Figure 4.

FOXK2 and FOXO3 do not generally compete for binding site occupancy. (A and D) Western blot analysis showing the level of endogenous FOXK2, FOXO3, and Flag-tagged FOXO3 protein in the U2OS-3xFLAG-FOXO3 cell line following treatment with 1 μg/ml doxycycline and either a non-targeting siRNA (con) or an siRNA targeting FOXK2 (A) or following treatment with or without 1 μg/ml doxycycline (dox) for 24 h (D). Protein expression was determined by immunoblotting (IB) with the indicated antibodies. (B, C and E) ChIP analysis of endogenous FOXK2 or Flag-tagged FOXO3 in U2OS-3xFLAG-FOXO3 (B and E) or U2OS (C) cells. (B) Cells were treated with doxycycline for 24 h and then LY294002 for 2 h before crosslinking and also treated with either a non-targeting siRNA (con) or a siRNA targeting FOXK2. ChIP experiments were performed with a FOXK2 (top) or Flag (FOXO3; bottom) antibody on genomic regions associated with the indicated loci. (C) ChIP experiments were performed with control IgG or a FOXK2 antibody on genomic regions associated with the indicated loci in cells left untreated or treated with LY294002 for 2 h before crosslinking. (E) Cells were treated with doxycycline for 24 h and then LY294002 for 2 h before crosslinking. ChIP experiments were performed with a Flag (FOXO3; top) or FOXK2 (bottom) antibody on genomic regions associated with the indicated loci. The error bars represent the standard deviations from two independent experiments. Where indicated, statistical significance is shown (* = P-value < 0.05 with a one tailed t-test (B) or two tailed t-test (E)).