Fig. 4.

VP56 decreases the phosphorylation of IRF7 mediated by TBK1. (A) VP56 has no significant effect on RLR protein expression. HEK 293T cells were seeded in 6-well plates overnight and transfected with 2 μg of HA-RLRs and 2 μg of empty vector or Myc-VP56 for 24 h. The WCLs were subjected to IB with the anti-HA, anti-Myc, and anti-β-actin Abs. (B and C) TBK1 mediates the phosphorylation of IRF3 and IRF7. HEK 293T cells were seeded into 6-well plates overnight and transfected with Flag-TBK1 and HA-IRF3/IRF7 (2 μg for each) for 24 h. The cell lysates (100 μg) were treated with or without CIP (10 U) for 40 min at 37 °C. The lysates were then detected by IB with anti-HA, anti-Flag and anti-β-actin Abs. (D and F) HEK 293T cells were seeded into 6-well plates overnight and co-transfected with 1 μg Flag-TBK1 or Flag-DrTBK1 plus 1 μg empty vector or HA-VP56, together with 1 μg Myc-IRF7 or Myc-DrIRF7 for 24 h. The lysates were then subjected to IB with anti-Myc, anti-Flag, anti-HA, and anti-β-actin Abs. (E and G)HEK 293T cells were seeded into 6-well plates overnight and co-transfected with 1 μg Flag-TBK1 or Flag-DrTBK1 plus various concentration of HA-VP56 (0.5 μg, or 1 μg, or 2 μg, empty vector was used to make up the rest), together with 1 μg Myc-IRF7 or Myc-DrIRF7 for 24 h. The lysates were then subjected to IB with anti-Myc, anti-Flag, anti-HA and anti-β-actin Abs. All experiments were repeated at least three times with similar results.