Figure 6.

PI-3 kinase/Akt pathway regulation. (A) Effect of the NADPH oxidase inhibitor DPI and exogenous H₂O₂ on PI-3 kinase/Akt pathway. The PI-3 kinase activity was measured in anti-phosphotyrosine immunoprecipitates from cells treated for 10 min with insulin, H₂O₂, insulin plus DPI, or insulin plus H₂O₂. Each bar represents the mean value ± SE of three independent experiments normalized to the value of unstimulated SK-N-SH cells. (B) Effect of DPI and LY294002 on the insulin-mediated translocation of PDK-1 and Akt activation in SK-N-SH cells. Membrane fractions and lysates were prepared from cells stimulated with insulin, insulin plus DPI, or insulin plus LY294002. Samples were fractionated (50 μg of protein) by 10% SDS-PAGE and transferred to nitrocellulose. PDK-1 and activated Akt were visualized using anti-PDK-1 and anti-phospho-Akt. The same blots were reprobed with anti-actin and anti-Akt, respectively, to confirm equal protein loading. (C) Effect of PI-3 kinase inhibitor on the Akt activation in response to exogenous H₂O₂. Proteins (50 μg) from cells stimulated with H₂O₂ or H₂O₂ plus LY294002, were fractionated on two 10% SDS-PAGs and transferred to nitrocellulose. One blot was used for visualizing phosphorylated Akt by using anti-phospho-Akt, and the other blot was used for visualizing Akt by using anti-Akt to confirm equal protein loading.