Figure 4

tBHP-induced PHLPP2 protein expression mediates site-specific Akt deactivation leading to Fyn kinase nuclear translocation and compromised Nrf2 signaling. Hepatocytes were treated with 250 μM tBHP for different time periods (15–180 min). (a) Immunoblot detection of key proteins involved in Nrf2 and Akt pathway. β-Actin was used as endogenous control to normalize the protein expression values. (b) Graph representing change in ratio of phosphorylated/total Akt and Fyn kinase during exposure to tBHP. Western blotting images of (c) PHLPP2 and mTORC2 in total cellular extract and (d) Nrf2, Fyn kinase, PHLPP2 and Akt(Ser473) in nuclear and cytosolic extracts. β-Actin lamin b were used as reference controls for cytosolic and nuclear extracts. (e) Immunofluorescence staining of hepatocytes for Fyn kinase (green) and Hoechst (blue) illustrating nuclear translocation of Fyn kinase upon tBHP exposure; (magnification × 40). The data are presented as mean±S.E. of at least three independent experiments. *P<0.05 compared with control