Fig. 3. Irf8 is association with Sting following DNA stimulation.
a Immunostaining of Irf8 (red) in BMDMs un-stimulated or stimulated with HSV-1 or IFN-γ (100 ng/ml) for 6 h. Scale bars, 50 μm. b Cell fractionation analysis of BMDMs infected with HSV-1 or SeV, or treated with IFN-γ (100 ng/mL) for the indicated times. The nuclear and cytoplasmic extracts were analyzed by immunoblotting with the indicated antibodies. c Immunoblot analysis of the indicated proteins in IRF8-KO or control THP-1 cells treated with 2′3′-cGAMP for the indicated times. d qPCR analysis of IFNB1, IFIT1 and CXCL10 mRNA in IRF8-KO or control THP-1 cells treated with 2′3′-cGAMP for 4 h. Data are presented as mean ± AD of one representative experiment, which was repeated for 2 times with similar results. n = 2 technical repeats. Data were analyzed by unpaired two-tailed Student’s t-test. e IRF8 interacts with STING in mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids for 24 h. Co-immunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. EV, empty vector. f Endogenous association of Irf8 and Sting in BMDM cells. The cells were left uninfected or infected with HSV-1 for the indicated times. Co-immunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. g Immunostaining analysis of IRF8 localization in HeLa transfected with IRF8-Flag (violet), STING-cherry (red) and the indicated GFP-tagged marker plasmids (green) for 24 h and then un-stimulated or stimulated with 2′3′-cGAMP for 4 h. GFP- Rab9 (ER marker), GFP-p58 (ERGIC marker), and GFP-GM130 (Golgi marker). Scale bars, 10 μm. h Immunostaining of p-StingS365 (green) and Irf8 (red) in murine lung fibroblasts stably transduced with Irf8 and infected with HSV-1 for 6 h or transfected with 2′3′-cGAMP and HSV120 for 4 h. Scale bars, 20 μm. Source data are provided as a Source data file.