Figure 4.

CSN6/COP1‐mediated FOXO4 ubiquitination requires physical interaction between COP1 and VP motif of FOXO4. A) The 293T cells were transfected with indicated HA‐FOXO4 VPAA plasmids. Cell lysates were immunoprecipitated with anti‐HA and immunoblotted with indicated antibodies. COP1's impacts on these FOXO4 VPAA plasmids are demonstrated. B) The 293T cells were cotransfected with indicated plasmids. Cells were treated with 5 µg mL⁻¹ MG132 (Sigma) for 6 h before harvesting. Cells were lysed in guanidine‐HCl containing buffer. The cell lysates were then pull down (PD) with nickel beads and immunoblotted with anti‐HA. C) 293T cells were co‐transfected with indicated plasmids. Cell lysates were immunoblotted with indicated antibodies. D) The 293T cells were cotransfected with indicated plasmids. Cells were treated with 5 µg mL⁻¹ MG132 (Sigma) for 6 h before harvesting. Cells were lysed in guanidine‐HCl containing buffer. The cell lysates were then pull down (PD) with nickel beads and immunoblotted with anti‐HA. E) Myc‐CSN6 overexpressing HCT116 cells were transfected with indicated plasmids. Cells were treated with cycloheximide (CHX) (100 µg mL⁻¹) for the indicated time. Cell lysates were immunoblotted with indicated antibodies. The turnover rate is shown. F) HCT116 cells were transfected with either HA‐FOXO4 or HA‐FOXO4 428VPAA mutant. Cells were treated with 100 ng mL⁻¹ EGF for the indicated minutes. Cell lysates were immunoblotted with indicated antibodies.