Figure 7.

Importin α5 binds NDV M protein by interacting with importin β1. (A) The subcellular localization of the fusion proteins in DF-1 cells. The indicated plasmids were transfected into DF-1 cells and then used for immunofluorescence aasay at 24 h post-transfection. DAPI was used to stain nuclei. Fluorescent images were obtained under a Nikon fluorescence microscope. Original magnification was 1 × 200. (B) Characterization of the interaction between NDV M protein and the cellular transport proteins by reciprocal co-immunoprecipitation assay. DF-1 cells transfected with the plasmids were lysed at 24 h post-transfection, and co-immunoprecipitation assay was performed using either anti-HA (upper panel) or anti-Myc (lower panel) antibodies. Immunoprecipitated proteins were detected by Western blotting using anti-Myc or anti-HA antibodies. (C) Identification of the interaction between importin α5 and M or importin β1 by pull-down assay. The purified GST-M or GST-importin β1 protein was immobilized on Gluthatione-Sepharose beads and then incubated with the purified His-importin α5. The bound proteins were eluted from the beads and examined by Western blotting. (D) Protein binding assay was used to identify the interaction among M, importin α5 and importin β1. Lane 1 was GST-M alone, lane 2 was GST-M plus His-importin α5, lane 3 was GST-M plus His-importin β1, lane 4 was GST-M plus His-importin α5 together with His-importin β1.