Figure 3

Inhibition of Akt suppresses Nrf2-regulated survival pathway by enhancing Fyn kinase activation and its nuclear translocation. LY294002 (10–50 μM) and PP1 (5-25 μM) were used to inhibit Akt and Fyn kinase, respectively. Western blotting analysis of (a) Nrf2 and its downstream targets NQO1 and HO1 and phosphorylated forms of Akt, GSK and Fyn kinase in total cellular extract and (b) Nrf2 and Fyn kinase in nuclear and cytosolic extracts. β-Actin was used as an endogenous control for total and cytosolic extracts while lamin b was used as a reference protein for nuclear extract. (c) Estimation of enzyme-linked immunosorbent assay-based Nrf2-ARE binding affinity in nuclear lysates obtained from hepatocytes treated with 30 μM LY294002 and 15 μM PP1 for 30 min. (d) Levels of ubiquitinated Nrf2 were analyzed by immunoprecipitating Nrf2 protein followed by western blotting detection with anti-ubiquitin antibody. (e) Nuclear translocation of Nrf2 upon Fyn kinase inhibition was assessed by immunofluorescence staining of hepatocytes for Nrf2 (green) and Hoechst (blue); (magnification × 40). (f) Nuclear translocation of Fyn kinase upon Akt inhibition was assessed by immunofluorescence staining of hepatocytes for Fyn kinase (green) and Hoechst (blue); (magnification × 40). The data are presented as mean±S.E. of at least three independent experiments. *P<0.05 compared with control