Figure 4

Akt and ERK phosphorylation induced by EGF in ovarian cancer cells and ERK is required for activation of cPLA ₂ . CAOV3 (A) and SKOV3 (B) cells were pretreated with AG1478 (10 μM) and/or WEB2086 (50 μM) for 1 h before exposure to EGF (10 ng/ml) for 10 min. Total protein was extracted and analyzed for phospho-Akt/total-Akt and phospho-ERK/total-ERK and was examined by immunoblot analysis. β-actin was used as a loading control. CAOV3 (C) and SKOV3 (D) cells were pretreated with the ERK inhibitor PD98059 (10 μM) and the Akt inhibitor LY294002 (10 μM) before exposure to EGF (10 ng/ml) for 10 min. Total protein was extracted and analyzed, and phospho-cPLA₂/total-cPLA₂ was examined by immunoblot analysis. β-actin was used as a loading control. For the immunofluorescence staining of phosphorylated cPLA₂ in CAOV3 (E) and SKOV3 (F) cells, after 10 min of incubation without any drug or with 10 ng/ml of EGF or with 10 ng/ml of EGF plus 10 μM of PD98059, cells were labeled with polyclonal antibody to phosphorylated cPLA₂ overnight, and then cells were incubated with fluoresent secondary antibody to phospho- cPLA₂ for 1 h and stained with DAPI for 10 min.