Fig. 5.

Trim25 directly interacts with RABV-P. (A) 293T cells were transfected with the plasmid vector pCAGGS or FLAG-Trim25. The cells were infected with CVS at an MOI of 1 at 12 h.p.t. and incubated for an additional 36 h. Then, the cells were lysed with NP-40 lysis buffer. Co-immunoprecipitation (Co-IP) was then performed using magnetic beads conjugated with antibodies against the FLAG-tag. Western blotting was applied to measure the expression of the indicated proteins. (B) 293T cells were infected with CVS at an MOI of 1 for 36 h, the cells were lysed, and IP was performed using magnetic beads conjugated with antibodies against the RABV-P protein. The expression of the indicated proteins was measured by Western blotting. (C) 293T cells were infected with CVS at an MOI of 1 for 36 h, fixed with 4% paraformaldehyde, stained with antibodies against Trim25, RABV-P, or DAPI, and observed under confocal fluorescence microscopy. The white arrow indicates the co-localization of Trim25 and RABV-P. Scale bar, 10 μm. (D) GST or GST-Trim25 was expressed in E. coli, and purified lysates containing GST or GST-Trim25 were incubated with HA-CVS-P or HA-CVS-N (expressed in 293T cells) to assess binding. (E) HA-CVS-P was expressed alone or co-expressed with FLAG-Trim25 or FLAG-E3-mut-Trim25 in 293T cells. Cell lysates were subjected to Co-IP and analyzed by Western blotting. Western blotting data are representative of at least two independent experiments. See also Fig. S3.