Figure 3.

FBXW8 interacts with the PDCoV N protein. (A, B) HEK-293T cells were co-transfected with Flag-tagged FBXW8 and HA-tagged PDCoV N plasmids for 24 h, and then the whole cell lysate (WCL) was purified by anti-Flag (A) or anti-HA (B) affinity gel for immunoblot analysis, respectively. (C) PDCoV N were cloned into pET28a-GST vectors and transformed into BL21(DE3) cells. The relationship between FBXW8 and PDCoV N protein was assessed with the use of the GST pull-down kit. (D) LLC-PK1 cells were uninfected or infected with PDCoV (MOI = 0.1) for 28h. Co-IP assay was performed with the endogenous FBXW8 and couples to protein A/G beads. (E) PK-15 cells were transfected with FBXW8 and PDCoV N plasmids, which were labeled with specific primary antibodies (Flag is red and HA is green). Cell nuclei were stained with DAPI (blue). Confocal immunofluorescence microscopy was used to visualize the results. (F) Schematic representation of FBXW8 fragments used for Co-IP analysis (up). HEK-293T cells were co-transfected with HA-PDCoV-N and truncated fragments of Flag-FBXW8. (G) Schematic representation of PDCoV N protein used for Co-IP analysis (up). HEK-293T cells were co-transfected with Flag-FBXW8 and truncated fragments of PDCoV N protein. The WCL was performed for Co-IP assay with anti-Flag affinity gel. The WCLs and immunoprecipitants (IB) were analyzed by Western blot.