Fig. 5.

GRFT interacts with JEV virions. (A) Pulldown assay. IMAC beads (50 μl) were preincubated with His-tagged GRFT (5 μg), washed, and incubated with the virus (MOI = 0.1) for 1.5 h. The beads were washed, and the JEV envelope protein was detected by western blot analysis using an anti-JEV monoclonal antibody. The JEV envelope protein could only be detected in the presence of GRFT. (B) Co-immunoprecipitation assay. GRFT (5 μg) was preincubated with the virus (MOI = 0.1) for 2 h. Protein G-Sepharose beads (50 μl) were incubated with rabbit anti-GRFT polyclonal antibodies for 2 h. The JEV/GRFT mixture was then incubated with the pretreated protein G-Sepharose beads for 2 h. The beads were washed, and JEV envelope protein was detected by western blot analysis using anti-JEV monoclonal antibody. The JEV envelope protein could only be co-immunoprecipitated in the presence of GRFT