(A) EGF (1h) treatment induced eNOS phosphorylation (Ser1177) in WT hepatocytes, arrow points to increased immunofluorescence. Western blotting for p-EGFR (Tyr1173), p-AKT (Ser473) and p-eNOS (Ser1177) expression in hepatocytes in response to EGF treatment (B) Time course (20 ng/ml) and (C) Dose-response (1h). Total EGFR, AKT and eNOS served as loading control. (D) Western blotting for p-EGFR (Tyr1173), p-AKT (Ser473) and p-eNOS (Ser1177) expression in WT hepatocytes pre-treated with DMSO (D), AG1478 (AG; 1 µM) or LY294002 (LY; 20 µM) for 30 min prior to EGF (E) treatment for 1h. (E) Western blotting for cyclin D1 and PCNA in hepatocyte pre-treated with inhibitors for 30 min prior to EGF treatment (24 h). Experiments were performed in triplicate with three independent hepatocyte isolations. Data represents mean+/− SD. *P<0.05, **p<0.01 vs WT untreated; #
p<0.05, ##
p<0.01 vs WT EGF treated.