Fig. 4
BinCARD1 interacts with NP. A, B A co-IP assay was performed to examine the interaction between NP and BinCARD1. HEK293T cells were transfected with plasmids expressing V5-WSNNP and Flag-BinCARD1 individually or in combination. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-NP mAb (A) or a mouse anti-Flag mAb (B) and subjected to western blotting with a rabbit anti-NP pAb or a rabbit anti-Flag pAb. C The BinCARD1-NP interaction did not rely on the RNA-binding activity of NP. HEK293T cells were transfected as described in A, B. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb, and the proteins were subjected to western blotting with a rabbit anti-NP pAb or a rabbit anti-Flag pAb. D A co-IP assay was performed to examine the interaction between NP and BinCARD1 in virus-infected cells. HEK293T cells were transfected with vectors to express Flag-BinCARD1, and at 36 h p.t., the cells were infected with WSN (H1N1) virus (MOI = 5) for 12 h. Cell lysate proteins were immunoprecipitated with a mouse anti-Flag mAb and then subjected to western blotting with a rabbit anti-NP pAb or a rabbit anti-Flag pAb. E Colocalization of NP and BinCARD1 was detected by confocal microscopy. A549 cells were transfected with plasmids expressing WSNNP and Flag-BinCARD1 individually or in combination. At 20 h p.t., the cells were stained with a mouse anti-NP mAb and a rabbit anti-Flag pAb, incubated with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (green), and visualized by confocal microscopy. F, G Co-IP assays were performed to examine the interaction between truncation mutants of NP and BinCARD1. HEK293T cells were transfected with the indicated constructs. At 36 h p.t., cell lysates were pulled down with glutathione magnetic beads. The bound proteins were eluted and subjected to western blotting with a rabbit anti-NP pAb, a rabbit anti-Flag pAb, or a rabbit anti-GST pAb
