Figure 5.

CpG ODN regulates phosphorylation and localization of FoxO3a through TLR9 activation. (A, B) Macrophages were starved serum for 12 h and treated with 3 µM CpG ODN for the indicated times. FoxO3a phosphorylation was then assessed by Western blotting using anti-FoxO3a or anti-phospho FoxO3a (T³² or S²⁵³) Ab. Representative blots are shown (A). Part of cells were separated into the cytosol and nuclear fractions, and analyzed for FoxO3a expression using anti-FoxO3a Ab or β-tubulin (B). (C) Cells were pretreated with TLR inhibitors (5 nM bafilomycin A1, 5 µg/ml CQ) for 1 h and stimulated with CpG ODN for 30 min. FoxO3a phosphorylation was then analyzed by Western blotting using anti-FoxO3a and anti-phospho FoxO3a Ab. (D) Cells were transfected with TLR9 siRNA or control siRNA for 24 h. Cells were incubated in free medium for 12 h, and stimulated with 3 µM CpG ODN for 30 min. FoxO3a phosphorylation was then analyzed by Western blotting using anti-FoxO3a and anti-phospho FoxO3a Ab.