FIGURE 4.

Knockdown of IRF3 or IRF8 regulate MyD88-mediated NF-κB activation. (A) HEK293 cells were cotransfected with the 0.4 μg of IRF3 or 0.4 μg of IRF8 plasmids with different concentrations of IRF3-shRNA (0.1, 0.2, or 0.4 μg) or IRF8-shRNA (0.1, 0.2, or 0.4 μg), respectively. After 24 h post transfection, IRF3 or IRF8 was determined by immunoblot assays. (B) HEK293 cells were seeded in 12-well plates and cotransfected with the IRF3 (0.2 or 0.4 μg) or IRF8 (0.2 or 0.4 μg) plasmids together with 0.4 μg of MyD88 and then 0.4 μg of IRF3 or IRF8 plasmid in the fourth well with 0.4 μg of IRF3-shRNA or IRF8-shRNA, respectively, together with 0.4 μg of MyD88. MyD88 was determined by immunoblot assays. (C) HEK293 cells were seeded in 12-well plates and cotransfected with 0.4 μg of IRF3 and IRF3-shRNA plasmids or IRF8 and IRF8-shRNA plasmids, respectively, together with 0.4 μg of MyD88. At 24 h post transfection, and cells were treated with CHX (100 μg/ml) and lysed at different time points. The expression of MyD88 was examined by immunoblot assays. (D) EPC cells were seeded in 24-well plates and cotransfected with 0.2 μg of IRF3 plasmid and IRF3-siRNA or 0.2 μg of IRF8 plasmid and IRF8-siRNA, respectively, together with 0.1 μg of MyD88 and 0.25 μg of NF-κB reporter gene, and the luciferase activity was measured at different times.*p < 0.05 versus the controls. All experiments were performed in at least three independent experiments.