Fig. 2

Validation of the interaction between ANXA2 and NS1. a Co-IP assay confirming the interaction between NS1 and ANXA2. A549 cells were transfected with HA-ANXA2 plasmid and infected with GD1322 24 h later. Cell lysates, harvested 36–48 h after infection, were subjected to IP and western blotting with anti-HA or anti-NS1 D7 antibodies. b Co-IP assay detecting the association between NS1 and endogenous ANXA2. A549 cells were infected with GD1322 and collected after 12 h. Next, cell lysates from infected or uninfected A549 cells were immunoprecipitated with an anti-ANXA2 rabbit antibody or the anti-NS1 D7 mouse antibody. After incubation with protein A/G-agarose beads, the Immunoprecipitation pellets were immunoblotted with the anti-NS1 D7 mouse antibody or the anti-ANXA2 rabbit antibody. c Pull down assay to verify that the ED of NS1 binds with ANXA2. Full-length NS1 and each functional domain were fused with GST and then incubated with uninfected A549 lysate. GST and ANXA2 were detected by western blotting. d Colocalization of ANXA2 and NS1 in A549 cells. HA-ANXA2 and FLAG-NS1 plasmids were co-transfected into A549 or 293 T cells. After incubation for 24 h, the cells were double-immunostained for FLAG-NS1 (green) and HA-ANXA2 (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue)