Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein
(A) U251 (lanes 2 and 4) and U251-UL23 cells (lanes 1 and 3) were treated with IFN-γ (1000 U/ml) and harvested at 36 hours post-treatment. The IFN-γ treated U251 cells were infected with HCMV TowneBAC (lanes 5 and 7) or ΔUL23 (lanes 6 and 8) (MOI = 1) at 12 hours post-treatment and harvested at 24 hours postinfection. The harvested cells were separated into nuclear and cytoplasmic fractions. Equivalent amounts of each fraction were analyzed by immunoblotting with anti-UL23, anti-Nmi, and anti-STAT1. The purity of the nuclear and cytoplasmic fractions was assayed by immunoblotting with anti-histone H1 and anti-actin, respectively. The membranes were reacted with antibodies and quantitated with a STORM840 PhosphorImager (GE Healthcare) or a Gel Documentation Station (BioRad, Hercules, CA) [60,64]. The protein levels of Nmi (B) and STAT1 (C) in the nuclei and cytoplasm of different cells that were mock-infected or infected with different viruses were quantified. The experiments were repeated three times. The standard deviation is indicated by the error bar.