Fig. 4

Both importin α3 major and minor sites are important for the HeV NiV W protein interactions. a Schematic of the mutations made in the W protein to test the binding contributions at the major and minor binding sites for importin α3. b Co-immunoprecipitation assay performed with Flag antibody on lysates of HEK293T cells expressing HA-tagged importin α3 and Flag-tagged HeV W, NiV W, and mutant W constructs as indicated. Western blots were performed for HA and Flag. WCL, whole cell lysate; IP, immunoprecipitation. pCAGGS denotes empty vector control. c HeV W mutations were tested against importin α3 using the ELISA assay. GST-HeV W and mutants were coated on 96-well plates, and binding of 6xHis-tagged importin α3 assessed using an anti-6xHis HRP antibody. Error bars show the S.E.M for three replicates. d Hela cells were transfected with indicated HA-tagged HeV and NiV W plasmids. Twenty-four hours post transfection, cells were fixed and stained with anti-HA antibody and Alexa Fluor 594 to visualise protein localisation. Image is representative. Scale bar depicted is 20 µm. The ratio of nuclear to cytoplasmic fluorescence (Fn/c) was determined for 50 cells for each construct, error bars represent the SEM