Figure 6.

SADS-CoV N protein disrupts the interaction between TBK1 and TRAF3. (A) HEK-293T cells were co-transfected with expression plasmids pCMV-FLAG-N and HA-TBK1 or mock-transfected for 24 h. The whole-cell lysates were collected and analyzed by Western blot with anti-FLAG, anti-HA, anti-TBK1, and anti-phosphorylated TBK1 monoclonal antibodies (MAbs), respectively. (B) HEK-293T cells were transfected with plasmids pCMV-FLAG-N with increasing quantities or mock-transfected for 24 h, and cells were treated or untreated with Sev for an additional 12 h. The whole-cell lysates were collected and immunoprecipitated with the protein A/G magnetic beads containing the anti-TBK1 antibody or normal rabbit IgG. (C) HEK-293T cells were co-transfected with expression plasmids MYC-TBK1 and HA-TRAF3 and pCMV-FLAG-N or empty vector for 28 h. The lysates were collected and immunoprecipitated with the protein A/G magnetic beads containing anti-TRAF3 antibody. ST cells were co-transfected with expression plasmids of porcine MYC-TBK1 and porcine HA-TRAF3 and pCMV-FLAG-N or empty vector for 28 h. The whole-cell lysates were collected and immunoprecipitated with the protein A/G magnetic beads containing anti-TBK1 antibody (D) or anti-TRAF3 (E). The protein expression levels were analyzed by Western blot with anti-FLAG, anti-TRAF3, anti-TBK1, anti-MYC, anti-HA, and anti-β-actin monoclonal antibodies (MAbs), respectively. Levels of TRAF3 or TBK1 after immunoprecipitation were quantitated densitometrically and corrected with TBK1 or TRAF3 in immunoprecipitation, and the ratio between the immunoprecipitated and co-immunoprecipitated proteins in untreated cells was set as 1.0.