Epstein-Barr Virus Interferes with the Amplification of IFNα Secretion by Activating Suppressor of Cytokine Signaling 3 in Primary Human Monocytes
Figure 3
Effect of EBV infection on the activation of the JAK/STAT pathway.
(A) Monocytes were incubated in the presence of IFNα (1000 U/ml) for 15 minutes and 20 hours. Following incubation (20 hours), cells were restimulated or not with IFNα for 15 minutes. The expression of phospho(p)STAT1, and phospho(p)STAT2 proteins was evaluated by Western blot analysis. Membranes were also probed with anti-Actin as a loading control. Densitometry was performed and represents fold protein induction (relative to non stimulated cells) ± std. dev. of experiments performed in duplicate. (B) Monocytes (5×106) were treated with IFNα (1000 U/ml) for 15 minutes, with EBV for 20 hours or were pre-incubated for 20 hours in the presence of EBV followed by a stimulation with IFNα for 15 minutes. The expression of phospho(p)Tyk2, phospho(p)JAK1, phospho(p)STAT1, and phospho(p)STAT2 proteins was evaluated by Western blot analysis. Membranes were also probed with anti-Tyk2, JAK1, STAT1 and STAT2 as a loading control. (C) Monocytes (2×106 cells) were transfected with 165 nM siRNA targeting SOCS1 or SOCS3 prior to EBV stimulation for 1 hour. Scramble siRNA was used as control. The expression of SOCS1 and SOCS3 was evaluated by Western blot analysis. Densitometry was performed and represents fold protein induction (relative to non-transfected cells) ± std. dev. of experiments performed in duplicate. (D) Monocytes (2×106 cells) were either left untransfected or were transfected with siRNA targeting SOCS1 or SOCS3 and stimulated as in (B). The expression of phospho(p)STAT1 and phospho(p)STAT2 proteins was evaluated by Western blot analysis. Membranes were also probed with anti-STAT1 and STAT2 as a loading control. Data are representative of three independent experiments. NS: non-stimulated; NT: non-transfected; SCR: scrambled siRNA.