Fig. 5

2N protein inhibits the interaction of TRIM25 and RIG-I through competitively binding to TRIM25. a HEK293T cells were transfected with the plasmids expressing 1 μg Flag-tagged SARS-CoV-2 N (Flag-2N) or empty-vector together with HA-TRIM25 in six-well plate. After 24 hpt, the cell lysates were subjected to anti-Flag immunoprecipitation (IP) and analyzed by immunoblot with anti-TRIM25, HA, and Flag antibodies, GAPDH was used as a loading control. b HEK293T cells were transfected with the indicated plasmids as in a for 24 h, the cell lysates were subjected to anti-HA IP and analyzed by immunoblot. c HEK293T cells were transfected with the plasmids expressing 0.25 μg GST-2N and Flag-TRIM25 in 24-well plate. After 24 hpt, the cells were subjected to immunofluorescence with anti-GST and Flag antibodies. d HEK293T cells were transfected with Flag-2N or empty vector for 24 h, the cell lysates were subjected to anti-Flag IP and analyzed by immunoblot with anti-TRIM25 and Flag antibodies. e HEK293T cells were transfected with Flag-RIG-I or empty vector and HA-TRIM25, together with or without 1 μg GST-2N plasmids in six-well plate. At 24 hpt, anti-Flag immunoprecipitates were analyzed by immunoblot. f HEK293T cells were transfected with Flag-TRIM25 and HA-RIG-I, together with an increasing concentration of GST-2N for 24 h in 6-well plate, anti-Flag immunoprecipitates were analyzed by immunoblot. g HEK293T cells were transfected with Flag-TRIM25 and HA-RIG-I, together with or without 0.25 μg 2N plasmids in 24-well plate. After 24 hpt, the transfected cells were subjected to immunofluorescence with anti-Flag and HA antibodies