The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

Figure 1

STK3 interacts with VP1. (a) HEK-293T cells were seeded in 10-cm dish, and the monolayer cells were transfected with 8 μg of a Myc- STK3 expressing plasmid (+), 8 μg of a FLAG-VP1 expressing plasmid (+), 8 μg of an empty FLAG vector (−), or 8 μg of an empty Myc vector (−). At 24 hpt, the cells were lysed, and the lysates were immunoprecipitated with mouse anti-Myc or mouse normal IgG antibodies and subjected to western blotting. The immunoprecipitated antibody-antigen complexes and whole-cell lysates were analyzed by immunoblotting using anti-FLAG, anti-Myc, or anti-β-actin antibodies. (b) Similar transfection and immunoprecipitation experiments were performed as described above. However, the lysates were immunoprecipitated with mouse anti-FLAG or mouse normal IgG antibodies and subjected to western blotting. (c) PK-15 cells were seeded in 10 cm dish, and the monolayer cells were transfected with 10 μg of a FLAG-VP1 expressing plasmid or 10 μg of an empty FLAG vector. The cells were lysed at 30 hpt. Cell lysates were immunoprecipitated with goat anti-STK3 and goat IgG antibodies and subjected to western blotting. The whole-cell lysates and immunoprecipitated antibody-antigen complexes were analyzed by immunoblotting using anti-STK3, anti-FLAG, or anti-β-actin antibodies. (d) Similar infection and immunoprecipitation experiments were performed as described above. However, the lysates were immunoprecipitated with mouse anti-FLAG or mouse normal IgG antibodies and subjected to western blotting. (e) HEK293T cells were seeded in Nunc glass bottom dishes, and the monolayer cells were transfected with 1.5 μg of a Myc-STK3 expressing plasmid, 1.5 μg of a FLAG-VP1 expressing plasmid, or 1.5 μg of an empty vector. At 24 hpt, the expression of Myc-STK3 and FLAG-VP1 was detected by an IFA analysis. Cells were double-immunostained for Myc-STK3 (red) and FLAG-VP1 (green); cellular nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

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