Fig. 7

The interaction domains of 2N protein and TRIM25. a HEK293T cells were transfected with the plasmids expressing GST-tagged full-length or truncated SARS-CoV-2 N (2N) protein, together with Flag-TRIM25 or Flag-vector. At 24 hpt, anti-Flag immunoprecipitates were analyzed by immunoblot with anti-GST and Flag antibodies. b, c HEK293T cells were transfected with an IFN-β promoter reporter and the full-length or truncated 2N plasmids, then treated with poly (I:C). At 24 hpt, luciferase activity was measured (b), and the mRNA expression of IFNA, IFNB1, and ISG15 were examined using qPCR (c). d Domain mapping of the SARS-CoV-2 N and TRIM25 association. e HEK293T cells were transfected with the indicated GST-tagged truncations of 2N and Flag-TRIM25 or Flag-vector. At 24 hpt, anti-Flag immunoprecipitates were analyzed by immunoblot. The arrow indicates the target protein band. f HEK293T cells were transfected with the GST-tagged truncations of 2N and Flag-TRIM25. At 24 hpt, the cell lysates were incubated using Glutathione Agarose to purify GST-S-2-N truncations. The purified proteins were analyzed by immunoblot. g Domain mapping of the association. h HEK293T cells were transfected with plasmids expressing GST-2N and Flag-TRIM25-SPRY domain. At 24 hpt, the cells were subjected to immunofluorescence analyses with anti-GST and Flag antibodies. i HEK293T cells were transfected with the plasmids expressing Flag-tagged full-length or truncated TRIM25, together with GST-2N. At 24 hpt, anti-Flag immunoprecipitates were analyzed by immunoblot. j HEK293T cells were transfected with the indicated plasmids. At 24 hpt, qPCR examined mRNA expression of SPRY and 2N. Results shown are the mean ± SD of at least three independent experiments