FIGURE 4.

HBsAg downregulates AKT phosphorylation via deactivation of PDPK1 and mTORC2. (A) Representative Western blot showing the effect of HBsAg on expression of total and phosphorylated forms of key molecules in the AKT signaling pathway in the HepG2-pcDNA3.1 and HepG2-pHBsAg cells. (B) Representative Western blot showing the effect of 4-PBA on expression of total and phosphorylated forms of key molecules in the AKT signaling pathway in the HepG2-pHBsAg cells. (C) Quantification of apoptotic cell fractions by annexin V/propidium iodide staining in the 4-PBA–pretreated HepG2-pcDNA3.1 and HepG2-pHBsAg cells 6 h after treatment with 1 μg/ml agonistic Fas CH11. Values are mean ± SD; n = 3. *p < 0.05. (D and E) Interaction between HBsAg and AKT, PDPK1, mTOR, and PTEN as determined by coimmunoprecipitation assay. FLAG-tagged HBsAg was immunoprecipitated with anti-FLAG Ab, and AKT, PDPK1, mTOR, or PTEN was detected by Western blotting with specific Abs. Reciprocal coimmunoprecipitation was carried out using AKT, PDPK1, mTOR, and PTEN Abs, and HBsAg was detected by Western blotting with anti-FLAG Ab. Normal IgG served as a control. (F) The effect of 4-PBA on interaction between mTOR and pmTOR (Ser²⁴⁸¹), Rictor, AKT, and pAKT (Ser⁴⁷³) in HepG2-pcDNA3.1 and HepG2-pHBsAg cells as determined by coimmunoprecipitation assay. mTOR was immunoprecipitated with the specific Ab, and pmTOR (Ser²⁴⁸¹), Rictor, AKT, or pAKT (Ser473) was detected by Western blotting with specific Abs.