FIGURE 2.

A, COP1 shRNA suppresses protein expression from COP1-transfected cells. HEK293 cells were transfected with FLAG-tagged COP1 and FLAG-tagged TRB1 expression vectors (200 ng/well) along with increasing amounts of COP1 shRNA expression vector (0, 100, and 200 ng, respectively). 48 h post-transfection, total cell lysates were prepared for Western blot analysis using anti FLAG-antibodies. B, COP1 shRNA suppresses endogenous COP1 expression. Fao cells were transduced with adenovirus expressing GFP or COP1 shRNA (shRNA). 36 h post-transduction the cells were treated with (or without) insulin (Ins) for 4 h, and total RNA was prepared for RT-PCR analysis using primers for COP1 (top) or 36B4 (bottom), respectively. C, COP1 suppresses FoxO1 protein expression in Fao hepatoma cells. Fao cells were transduced with GFP (–)-, COP1 (WT)-, and COP1 shRNA (shRNA)-expressing adenoviruses. 24 h post-transduction cells were serum-deprived overnight followed by 4 h of 100 nm insulin treatment (+). Total cell lysates prepared after insulin treatment were subjected to Western blot analysis using anti-COP1 (i), anti-FoxO1 (ii), anti-C/EBPα (iii), and anti-C/EBPβ (iv), antibodies, respectively. CTL, control. D, the experiments are carried out as in C, except COP1 expressing pMIGR-1 retroviruses were used. E and F, quantification of FoxO1 and C/EBPβ expression in C and D. The levels of FoxO1 or C/EBPβ were set as 1 under control condition (no effector expression and insulin treatment). Each bar represents the means ± S.D. (n = 3). The p values were related to the control conditions. *, p < 0.02. **, p < 0.012. #, p < 0.023.