Figure 5
Figure 5. IRF8 cooperates with IRF3 to regulate IFN-β transcription. (A) The genomic sequence of human IFN-β from −79 bp to −58 bp. The IRF3- and IRF8-binding sites are labeled in blue and red, respectively. (B) THP-1 cells were induced with LPS for 2 hours before total lysates were harvested and immunoprecipitation was performed. Western blotting of immunoprecipitated proteins is shown. Similar results were obtained in 3 independent experiments. (C) Combined knockdown of IRF8 and IRF3 using siRNA results in a pronounced reduction of IFN-β transcription in primary monocytes. Monocytes were transfected with control siRNA, IRF8 siRNA, IRF3 siRNA, or both IRF8 and IRF3 siRNAs. mRNA levels of IFN-β in LPS simulated-monocytes were determined by real-time PCR. Similar results were obtained in 3 independent experiments with monocytes from 3 different donors, one of which is shown. (D) 32Dcl3 cells were transfected with control, IRF3 plasmid, IRF8 plasmid, or both IRF8 and IRF3 plasmids, and then cells were stimulated with Sendai virus for 2 hours to activate expression of an IFN-β promoter luciferase. Values are mean plus or minus SD from 3 independent experiments. (E) EMSA was performed with siRNA-transfected THP-1 nuclear extract. Lane 1 indicates free probe; lanes 2 and 3, nuclear extract from LPS-stimulated THP-1 cells transfected with siRNA (lane 2 indicates control siRNA; and lane 3, IRF8 siRNA). (F) EMSA was performed with LPS stimulated THP-1 nuclear extracts. WT(−83 to −55) probe and M-70(−83 to −55) probes were used. Similar results were obtained in 3 independent experiments, one of which is shown. (G) IRF8–2HA mutants overexpressed in HEK293 cells with IRF3–5D-2FLAG or IRF3–5D were immunoprecipitated with anti-FLAG antibody. Western blot was performed with anti-HA antibody. (H) IRF3–5D-2FLAG and IRF3–5D mutants were overexpressed together with IRF8–2HA in HEK293 cells and immunoprecipitated with anti-FLAG antibody or antimouse IgG. Western blot was performed with anti-HA antibody. (E-H) Similar results were obtained in 3 independent experiments, one of which is shown.

IRF8 cooperates with IRF3 to regulate IFN-β transcription. (A) The genomic sequence of human IFN-β from −79 bp to −58 bp. The IRF3- and IRF8-binding sites are labeled in blue and red, respectively. (B) THP-1 cells were induced with LPS for 2 hours before total lysates were harvested and immunoprecipitation was performed. Western blotting of immunoprecipitated proteins is shown. Similar results were obtained in 3 independent experiments. (C) Combined knockdown of IRF8 and IRF3 using siRNA results in a pronounced reduction of IFN-β transcription in primary monocytes. Monocytes were transfected with control siRNA, IRF8 siRNA, IRF3 siRNA, or both IRF8 and IRF3 siRNAs. mRNA levels of IFN-β in LPS simulated-monocytes were determined by real-time PCR. Similar results were obtained in 3 independent experiments with monocytes from 3 different donors, one of which is shown. (D) 32Dcl3 cells were transfected with control, IRF3 plasmid, IRF8 plasmid, or both IRF8 and IRF3 plasmids, and then cells were stimulated with Sendai virus for 2 hours to activate expression of an IFN-β promoter luciferase. Values are mean plus or minus SD from 3 independent experiments. (E) EMSA was performed with siRNA-transfected THP-1 nuclear extract. Lane 1 indicates free probe; lanes 2 and 3, nuclear extract from LPS-stimulated THP-1 cells transfected with siRNA (lane 2 indicates control siRNA; and lane 3, IRF8 siRNA). (F) EMSA was performed with LPS stimulated THP-1 nuclear extracts. WT(−83 to −55) probe and M-70(−83 to −55) probes were used. Similar results were obtained in 3 independent experiments, one of which is shown. (G) IRF8–2HA mutants overexpressed in HEK293 cells with IRF3–5D-2FLAG or IRF3–5D were immunoprecipitated with anti-FLAG antibody. Western blot was performed with anti-HA antibody. (H) IRF3–5D-2FLAG and IRF3–5D mutants were overexpressed together with IRF8–2HA in HEK293 cells and immunoprecipitated with anti-FLAG antibody or antimouse IgG. Western blot was performed with anti-HA antibody. (E-H) Similar results were obtained in 3 independent experiments, one of which is shown.

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