Fig. 5.

VP56 promotes K48-linked ubiquitination and degradation of IRF7. (A) HEK 293T cells were seeded in 6-well plates overnight and transfected with 1 μg HA-IRF7 and 1 μg empty vector, or Myc-VP56 and 1 μg Flag-TBK1. At 18 h post-transfection, the cells were treated with the indicated inhibitors for 6 h prior to being harvested for IB analysis of WCLs with the anti-HA, anti-Myc, anti-Flag, and anti-β-actin Abs. (B) EPC cells were seeded in 10-cm² dishes and transfected with 4 μg Flag-TBK1, 4 μg Myc-IRF7, 4 μg HA-VP56 or empty vector, and 2 μg HA-Ub. At 18 h post transfection, the cells were treated with DMSO or MG132 for 6 h. Cell lysates were IP with anti-Myc-affinity gels. Then the immunoprecipitates and WCLs were analyzed by IB with the Abs indicated. (C) EPC cells were seeded in 10-cm² dishes and transfected with 4 μg Flag-TBK1, 4 μg Myc-IRF7, 4 μg HA-VP56 or empty vector, and 2 μg HA-Ub-K48O or HA-Ub-K63O. At 18 h post-transfection, the cells were treated with MG132 for 6 h. Cell lysates were IP with anti-Myc-affinity gels. Then the immunoprecipitates and WCLs were analyzed by IB with the indicated Abs. All experiments were repeated for at least three times with similar results.