FIG. 3.

Role of PI(3)K pathway in PKB/Akt phosphorylation and herpesvirus gene expression. (A) Replicate cultures of HEp-2 cells were mock infected or exposed to 10 PFU of HSV-1(F) or ΔU_S3 mutant virus per cell; after 7 h, cells were exposed to EGF (100 ng/ml) for 20 min, harvested, solubilized, and normalized for protein content. Equal amounts of protein lysates (120 μg/lane) were subjected to electrophoresis on a denaturing gel, transferred to a nitrocellulose sheet, and reacted with the indicated antibodies. (B) HEp-2 cells were mock infected or incubated with HSV-1(F) for 1 h and then exposed to the PI(3)K inhibitor LY294002 (75 μM) for 2 h. The cells were then treated with EGF (100 ng/ml) for an additional 30 min, harvested, and solubilized. Equal amounts of protein lysates (120 μg/lane) were subjected to electrophoresis and immunoblotting with the indicated antibodies. (C) Replicate cultures of HEp-2 cells were mock infected or exposed to 10 PFU of HSV-1(F) or ΔU_S3 or ΔICP4 mutant virus per cell and then exposed to LY294002 (75 μM) solubilized in dimethyl sulfoxide or to an equivalent amount of dimethyl sulfoxide added to the medium. The cells were harvested at 3.5 h after the infection and processed as described above. While 120 μg/lane of protein lysates was used for immunoblotting with antibodies against total and phosphorylated PKB/Akt, 60 μg/lane was used for immunoblotting with antibodies against viral proteins.