Fig. 4. Effect of HQ on AKT activation in IRF3 pathway.

(A) RAW264.7 cells (1×10⁶ cells/ml) pretreated with either HQ (50 µM) or vehicle for 30 min were incubated with LPS (1.0 µg/ml) for the indicated time. Phosphorylated and total levels of TBK1 and AKT in total cell lysates were determined by Western blot analysis. (B) RAW264.7 cells (1×10⁶ cells/ml) transfected with HA-AKT (1.0 µg/ml) for 24 h were treated with the indicated doses of HQ for another 24 h, and phosphorylated and total levels of IRF-3 and HA in total cell lysates were determined by Western blot analysis. (C) RAW264.7 cells (1×10⁶ cells/ml) were transfected with either HA-AKT (1.0 µg/ml) or HA-AKT C310A (1.0 µg/ml) for 48 h. Phosphorylated and total levels of IRF-3, AKT, and HA in total cell lysates were determined by Western blot analysis. (D) RAW264.7 cells (1×10⁶ cells/ml) transfected with either HA-AKT1 (1.0 µg/ml), HA-AKT2 (1.0 µg/ml), or HA-AKT3 (1.0 µg/ml) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Phosphorylated and total levels of IRF-3, AKT, and HA in the total cell lysates were determined by Western blot analysis. (E) HEK293 cells (1×10⁶ cells/ml) cotransfected with TBK1 (1.0 µg/ml), IRF-3-Luc (1.0 µg/ml), β-gal (0.1 µg/ml), and HA-AKT1 (1.0 µg/ml), HA-AKT2 (1.0 µg/ml), HA-AKT3 (1.0 µg/ml), or HA-AKT KD (kinase domain deletion mutant) for 24 h were treated with either HQ (50 µM) or vehicle for another 24 h. Luciferase activity was measured with a luminometer. All luciferase reporter gene activities were normalized to β-galactosidase activity. ^*p<0.05, ^**p<0.01 compared to the control.