Figure 2.

Interaction of IBDV VP4 with CypA. (a) CO-IP of HA-VP4 and CypA-Flag. PCAH-VP4 and pCAF-CypA were transfected into HEK293T cells together or alone. At 48 h after transfection, the cells were lysed for immunoprecipitation (IP) directed by anti-HA mAb. The proteins were detected by western blotting with anti-HA and anti-Flag mAb. Coexpressing HA-VP4 and CypA-Flag can form a complex, whereas neither single HA-VP4 nor CypA-Flag alone could. (b) GST pull-down assay. The soluble protein GST-CypA expressed in E. coli BL21 and GST alone were conjugated to glutathione beads and incubated with HEK293T cell lysates expressing the HA-VP4 protein. HA-VP4 was first detected with anti-HA mAb by western blot. GST-CypA or GST was then detected with anti-GST polyclonal antibodies. Cell lysates containing HA-VP4 were used as a control to show VP4 in the last lane. ∗ indicates nonspecific binding. (c) Colocalization of VP4 and CypA. DF-1 cells were cotransfected with pCAH-VP4 and pCAF-CypA. A single transfection was used as a control. At 48 h after transfection, the cells were fixed and subjected to indirect immunofluorescence assays to detect VP4 (red) and CypA (green) with anti-Flag mAb and anti-HA polyclonal antibody. DAPI (blue) was used to indicate the nucleus.