Figure 11

HCV infection down-regulate IFNAR1 and induces defective Jak-Stat signaling through ER-stress mechanisms. (A) Cured S-5/15 Huh-7 cells were infected with cell culture derived HCV. Infected and uninfected cells were collected at different time points (0 to 96 hrs) and examined for mature form of IFNAR1 protein by Western blot analysis. (B) The cellular expression of IFNAR1 in the uninfected and infected Huh-7 cells was examined by Flow analysis at different time (0 to 240 hrs). The percentage of positive cells at each time point indicates the cells having the IFNAR1 expression. The peak shifts with time. (C) The phosphorylation of Stat1 and Stat2 protein in the HCV transfected Huh-7 cells was examined by Western blot analysis. The cells were treated with IFN-α for 30 minutes prior to lysis for detection of phosphoproteins. The beta actin in all the blots was used as loading control. (D) Jak-Stat signaling of HCV transfected Huh-7 cells was examined at different time points using a Firefly Luciferase activity of interferon-beta promoter. (E) HCV replicating Huh-7 cells were collected at different time intervals (0 to 96 hrs), protein extracts were examined for the activation of ER-stress response using panel of monoclonal and polyclonal antibodies to IRE1-α, Bip, PERK and phospho EIF2-α. Beta actin level were measured to assure, that equal amounts of proteins were loaded onto the SDS-PAGE gel are used in the Western blotting.