Figure 4.

Analysis of the induction of IRGs by DB addition to infected cells and the relevance of the IFN signaling pathway. (a) Representative immunoblot analysis of 5 × 10⁵ HFF incubated with increasing amounts of UV-inactivated DBs derived from the HCMV strain TR-∆GFP. Cell lysates were prepared one day after DB application and subjected to SDS-PAGE and immunoblot analyses. Untreated cells (−) served as the control. The membranes were probed with antibodies against MX1, IFIT3 and ISG15. An antibody directed against the viral pp65 protein was used to confirm DB internalization. Tubulin served as the sample loading control. (b) HFFs were left uninfected, were infected with TB40/E (moi 0.5) or were pre-treated with 10 µg of UV-irradiated DBs for 2 h before infection with TB40/E (moi 0.5). IRG and viral pp65 and UL44 expression levels were measured up to 3 d.p.i by Western blotting. Tubulin was used as loading control. (c) Representative immunoblot analysis of MX1, IFIT3, and ISG15 expression in HFFs upon JAK inhibitor I treatment, compared to the untreated control cells. HFFs were incubated in 5% MEM medium in the presence or absence of JAK inhibitor I (20 μM/mL) for one hour. Afterwards, the cells were infected with the strain TB40/E or infected and simultaneously co-incubated with 10 µg of UV-irradiated DBs. Each sample was further co-treated with JAK inhibitor I for 24 h. Addition of IFN-β (100 U/mL) was used to control the effects of the inhibitor on the JAK-STAT signaling cascade. Pp65 was used as a DB internalization control.