Figure 2.

CSN6 increases FOXO4 turnover rate and enhances FOXO4 ubiquitination. A) HCT116 cell lysates were immunoprecipitated with either control rabbit IgG, CSN6, or FOXO4 antibodies followed by immunoblotting with indicated antibodies. B) CSN6 full length wild‐type, N‐terminal (aa 1‐184) or C‐terminal (aa 185‐327) was transfected into 293T cells. Cell lysates were immunoprecipitated with anti‐HA and immunoblotted with anti‐Flag for binding studies. C) SW480 cells transfected with the indicated plasmids were treated with cycloheximide (CHX) (100 µg mL⁻¹) for the indicated hours. Cell lysates were immunoblotted with indicated antibodies. The turnover rate of FOXO4 is shown. D) SW480 cells transfected with indicated plasmids were treated with or without proteasome inhibitor MG132. Lysates were immunoblotted with indicated antibodies. E) Cells transfected with the indicated CSN6‐shRNA (left, HCT116 cells) or CSN6 plasmids (right, 293T cells) were treated with 5 µg mL⁻¹ MG132 (Sigma) for 6 h before harvesting. Cells were lysed in guanidine‐HCl containing buffer and cell lysates were then pull down (PD) with nickel beads and immunoblotted with indicated antibodies. F) The 293T cells were cotransfected with indicated plasmids. Cells were treated with 5 µg mL⁻¹ MG132 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.