Figure 3
TNF-α activates ISRE in a STAT1 independent manner.
(A) Western blot analysis of total STAT1 and phosphorylated STAT1 (Y701P) protein levels under the treatment of TNF-α (100 ng/ml), IFN-α (1000IU). (B) Same as (A) for the detection of total STAT2 and phosphorylated STAT2 (Y690P) protein levels under the treatment of TNF-α , IFN-α . (C) Confocal microscopy analysis of phosphorylated STAT1 (Y701P) localization in Huh7 cells treated with IFN-α or TNF-α . STAT1 was phosphorylated and translocated to the nucleus upon IFN-α , but not TNF-α treatment. Phosphorylated STAT1 (Y701P) antibody (green). Nuclei were visualized by DAPI (blue). (D) Same as (C) for the detection and localization of phosphorylated STAT2 (Y690P). (E) qRT-PCR confirmed the successful STAT1 knockdown by lentiviral shRNA vectors in the Huh7 based ISRE luciferase reporter cells. (F) Western blot analysis confirmed the successful knockdown of STAT1 by lentiviral shRNA vectors in the Huh7 based ISRE luciferase reporter cells. (G) STAT1 knockdown had no significant influence on TNF-α induced ISRE-related luciferase activation as measured at 3 different time points (n = 3 independent experiments with 2–3 replicates each). (H) STAT1 knockdown exerts no effect on TNF-α induced ISG expression as measured by qRT-PCR (n = 3).
