Fig. 5.

PDCoV N protein promoted the degradation of poIRF7. (A) PK-15 cells were transfected with 200 ng of Flag-poIRF7 with increasing amounts of HA-PDCoV-N plasmid (200 ng, 400 ng, or 800 ng) for 24 h. (B) PK-15 cells were transfected with 1 μg HA-PDCoV-N or empty vector plasmid for 24 h. (C) LLC-PK1 cells were mock-infected or infected by PDCoV at MOI of 0.5. The whole cell proteins were extracted at the indicated post-infection hours. (B, C) The whole cell lysates were analyzed through Western blot with the commercialized hIRF7 or hIRF3 antibody. (D) PK-15 cells were co-transfected with 300 ng Flag-tagged poIRF7 and 700 ng HA-tagged PDCoV-N or empty vector plasmids. After 24 h, cells were treated with protein synthesis inhibitor cycloheximide (CHX) 200 μg/ml for the indicated time before analysis of the protein levels by Western blot. (E) PK-15 cells were co-transfected with 300 ng Flag-tagged IRF7 and 700 ng HA-tagged PDCoV-N or empty vector plasmids for 18 h. Then, the cells were treated with MG132 (25 μM) or DMSO as a control for 6 h before harvesting. The ex-pression level of poIRF7, which was normalized to the β-actin, was determined by densitometry analysis with ImageJ software. All of the experiments were performed independently three times.