FIG. 4.
FoxO6 subcellular distribution, DNA binding activity, and interaction with CRM-1. A: Immunohistochemistry. HepG2 cells pretransduced with 50 plaque forming units (pfu)/cell of FoxO6 vector (a and b) or FoxO1 vector (c and d) were serum-starved for 6 h, followed by incubation in the absence or presence of insulin (100 nmol/L) for 30 min. Cells were immunostained using rabbit anti-FoxO6 (a, b) or anti-FoxO1 (c, d) antibodies, followed by anti-rabbit IgG conjugated with fluorescein isothiocyanate (green). Bar = 25 μm. B: Immunoblot. HepG2 cells were transduced with 50 pfu/cell of FoxO6 vector in the absence or presence of Adv-Akt-CA vector (50 pfu/cell) expressing Akt-CA. After 24-h incubation, cells were harvested for the preparation of nuclear and cytoplasmic fractions, which were subjected to anti-FoxO6 immunoblot assay. C: Chromatin immunoprecipitation (ChIP) analysis of liver. C57BL/6J male mice (aged 10 weeks) were fasted for 16 h, followed by an injection of insulin (2 units/kg i.p.; n = 3) or phosphate-buffered saline (PBS; n = 3). Mice were killed 20 min after insulin injection, and aliquots of liver tissue (20 mg) underwent ChIP assay using rabbit anti-FoxO6 antibody for determining FoxO6 association with G6Pase promoter DNA. D: The amount of G6Pase promoter DNA bound by FoxO6 relative to input control DNA was determined in the liver of PBS- and insulin-treated mice. In addition, HepG2 cells were cotransduced with FoxO1 and FoxO6 vectors at a fixed dose (50 pfu/cell). *P < 0.005 vs. control by ANOVA. After a 24-h incubation, cells were collected for the preparation of total protein lysates, which were subjected to immunoprecipitation using anti–14-3-3 (E), anti-FoxO1 (F), anti–CRM-1 (G), or anti-FoxO6 antibody (H). The control antibody was anti–β-galactosidase IgG. The immunoprecipitates were analyzed for the presence of FoxO1, FoxO6, 14-3-3, or CRM-1. IB, immunoblotting; IP, immunoprecipitation; MW, molecular weight; bp, base pair. (A high-quality digital representation of this figure is available in the online issue.)