Skip to main content
. 2017 Mar 24;8:350. doi: 10.3389/fimmu.2017.00350

Figure 8.

Figure 8

The mechanism of H1C regulating interferon-β (IFN-β). (A) Analysis of the effects of H1C and its mutants on IRF3 and IRF3 phosphorylation. Human embryonic kidney 293 T (HEK293T) cells were overexpressed with H1C or its mutants and infected with Sendai virus (Sev) for an additional 12 h. IRF3 and IRF3 phosphorylation levels were then detected by western blotting using anti-IRF3 (sc-33642, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-IRF3-P antibody (Cat.#07-582-I, Millipore, Billerica, MA, USA). (B) Analysis of the effect of H1C on IRF3 nuclear transportation. HEK293T cells were treated as described above, and the cytoplasm and nuclear fractions were separated. IRF3 phosphorylation levels were then detected. (C) Detection of H1C–IRF3-P interactions; HEK293T cells co-transfected with HA-IRF3 and Flag-H1C or its mutants were infected with Sev for 12 h, and the co-immunoprecipitation (Co-IP) experiments were performed using anti-flag antibody and analyzed by western blotting using the anti-IRF3-P antibody to detect its interaction. (D) Investigation of co-localizations between H1C and IRF3-P. A549 cells cultured on slides were co-transfected with HA-IRF3 and Flag-H1C or its mutants and stimulated with poly (I:C) for 6 h, and immunofluorescence confocal microscopy was performed as previously described using anti-Flag mouse monoclonal antibody and anti-HA rabbit polyclonal antibody followed by immunostaining with FITC-labeled goat anti-mouse secondary antibody and Cy3-labeled goat anti-rabbit antibody. (E) Chromatin immunoprecipitation and quantitation PCR was performed to detect the effects of H1C on the capacity of IRF3 binding to the IFN-β promoter. HEK293T cells were co-transfected with HA-IRF3, and Flag-H1C or H1C mutants and chromatin immunoprecipitation assay were performed using am anti-HA antibody; the negative control was performed using the mouse IgG antibody, and the positive control was conducted using the anti-poly II antibody (*p < 0.05, the data were generated from one of three independent experiments).