FIG 1.

SARS-CoV N protein interacts with TRIM25. (A) Cell extracts prepared from 293T cells transfected with Flag-SARS N protein (Flag-N) or Flag vector were subjected to anti-Flag immunoprecipitation. The immunoprecipitates were resolved using SDS-PAGE electrophoresis and Coomassie blue staining. (B) The stained bands marked by arrows in Fig. 1A were digested with trypsin and analyzed by LC-MS/MS. Two peptides (SDLGAVAKGLSGELGTR and EPEELGK) matching the TRIM25 protein sequence were identified. (C and D) 293T cells transfected with the indicated plasmids were infected with or without SeV (C), and the cell lysates were subjected to anti-Flag immunoprecipitation (IP). The immunoprecipitates were analyzed by immunoblotting (IB) with anti-TRIM25 (C) or anti-Myc (D). (E) HeLa cells transfected with GFP-N were fixed with 4% paraformaldehyde and incubated with anti-TRIM25 and anti-GFP antibodies. An in situ PLA assay was conducted as described and imaged with a confocal microscope at ×100 magnification. Red dots indicate the interaction.