Figure 2.

Modulation of AKT/FOXO by H₂O₂ in cerebellar neurons. (A) In the presence of H₂O₂, IGF-I–induced phosphoAKT (pAKT, 15 min after IGF-I) was significantly blocked. Levels of total AKT were measured as loading control. **p < 0.01 versus all other groups; n = 5. (B) Nuclear translocation of pAKT after IGF-I was blocked by H₂O₂. NeuN levels were determined as nuclear fraction control (n = 3). (C) In response to IGF-I levels of FOXO3 in the nucleus 4 h later were reduced, but this effect was blocked by H₂O₂. NeuN was used as a marker of the nuclear fraction; n = 3. (D) The opposite effect is observed with cytoplasmic levels of FOXO3, which were increased 4 h after IGF-I, and addition of H₂O₂ counteracted this effect. β-actin was measured as a marker of the cytosolic fraction (n = 4). (E) Transcriptional activity of FOXO was significantly attenuated by IGF-I after 8 h, but addition of H₂O₂ greatly enhanced it; n = 3. **p < 0.01 versus IGF-I–treated cells. (F) Neurons cotransfected with GFP and dominant negative (DN)-FOXO3 were protected against the deleterious effects of H₂O₂ compared with GFP-transfected neurons (controls), were a 40% drop in the number of GFP⁺ cells was observed 6 h after treatment with H₂O_2, regardless of the presence or absence of IGF-1. ***p < 0.001, and **p < 0.01 versus controls (n = 3).