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. 2017 Dec 30;515:165–175. doi: 10.1016/j.virol.2017.12.028

Fig. 3.

Fig. 3

Interaction of proteins 8b and 8ab with IRF3. a. Schematic diagram showing the functional domains of IRF3 and various IRF3 deletion constructs used. The N-terminal DNA-binding domain (DBD), nuclear export signal (NES), proline-rich region (Pro), the IRF association domain (IAD) and the C-terminal repeat domain (RD) are shown. Fragments that immunoprecipitated with protein 8b are denoted with (+). b. Co-immunoprecipitation of Myc-tagged IRF3 with Flag-tagged 8b and 8ab, respectively. Cos7 cells were infected with the recombinant vaccinia/T7 virus at an MOI of approximately 5 per cell. After incubation for 1, cells were transfected with pMyc-IRF3, pFlag-8b, pMyc-IRF3+pFlag-8b, pFlag-8ab and pMyc-IRF3+pFlag-8ab, respectively. Cells were harvested at 18 h post-transfection, lysates prepared, and subjected to immunoprecipitation with either the Myc antibody-conjugated agarose beads (top two panels) or the Flag-antibody-conjugated beads (bottom two panels). The precipitates were separated on SDS-PAGE and analyzed by Western blot with either anti-Myc (top and bottom panels) or anti-Flag (2nd and 3rd panels) antibodies. c. Pull down of the endogenous IRF3 protein with protein 8b. H1299 cells were infected with wtIBV, rIBV8b and rIBV8ab, respectively. Cells were lysed 20 h post-infection and immunoprecipitated with either polyclonal antibodies raised against SARS-CoV 8b or control IgG antibodies. The precipitates were probed with antibodies against the full-length IRF3. d. Co-immunoprecipitation analysis of the Flag-tagged protein 8b co-expressed with the untagged IRF3 from 1–193, 1–375 and 1–427 (full-length). H1299 cells were infected with the recombinant vaccinia/T7 virus at an MOI of approximately 5 per cell. Immunoprecipitation was performed as described above. Total lysates and the precipitates were separated on SDS-PAGE and analyzed by Western blot with either anti-Myc or anti-Flag antibodies. e. Co-immunoprecipitation analysis of the Flag-tagged protein 8b co-expressed with the Myc-tagged IRF3 from 1–133, 134–240 and 134–427. H1299 cells were infected with the recombinant vaccinia/T7 virus at an MOI of approximately 5 per cell. Immunoprecipitation was performed as described above. Total lysates and the precipitates were separated on SDS-PAGE and analyzed by Western blot with either anti-Myc or anti-Flag antibodies.