FIG. 2.

PI3K and Akt are activated in cells expressing the complete HCV polyprotein. (a) PI3K assay. HepG2 cells were cotransduced with BactTA and BacH77Δ3′UTR, incubated in the presence (lane 1) or absence (lane 2) of tetracycline, and harvested at 24 h. PI3K was immunoprecipitated and subjected to an in vitro kinase assay in the presence of [γ-³²P]ATP and phosphatidylinositol as substrate as described previously (45). Reaction products were separated by thin-layer chromatography and detected by autoradiography (panel i). The position of the phosphorylated product is indicated by an arrowhead. Quantification was performed with a phosphorimager (Fuji, FLA-5000). An aliquot of the immunoprecipitation was processed for immunoblot analysis to confirm equal levels of p85 (panel ii). Panel iii shows an immunoblot of lysates for NS5A expression. (b) Akt phosphorylation assay. Lysates from HepG2 cells cotransduced with BactTA and BacH77Δ3′UTR were processed for immunoblotting with antibodies to the following proteins: for panel i, Akt phosphorylated on Ser473; for panel ii, a phosphorylation state-independent antibody to detect total levels of Akt; for panel iii, PDK1 phosphorylated on Ser 241; and for panel iv, NS5A. Samples in lanes 3 and 4 were treated with 50 μM Ly294002 for 16 h prior to harvest.