Fig. 2: IRF1 is activated upon interaction with STING.
From: T cell fate following Salmonella infection is determined by a STING-IRF1 signaling axis in mice
a Differential gene expression analysis of IRFs in wild-type and Tmem173−/− BM-DCs after stimulation by c-di-GMP (50 μg/ml) for 18 h. b Real-time PCR analysis of Irf1, Irf3, and Irf7 mRNA expressions in BM-DCs after stimulation with c-di-GMP for 4 and 18 h. Results are presented relative to normalized expression of the 18S ribosomal RNA. n = 4. ***p < 0.001. (Two-way ANOVA followed by Tukey’s multiple-comparisons test.) c BMDCs from wild-type, Tmem173−/−, and Irf1−/− (1 × 106 per time point) were stimulated with 50 μg/ml of c-di-GMP as indicated. Cells were lysed and nuclear compartments were analyzed by Western blotting with indicated antibodies (Abs). IRF-1 was visualized first; the membrane was then stripped and re-probed with anti-Lamin A/C. d Human STING variants and IRF1 were co-expressed in HEK239T cells and either STING or IRF1 immunoprecipitated, and detected with specific antibodies by Western blotting. Expression levels of STING or IRF1 were validated in total cell extracts by SDS–PAGE and immunoblotting. e Immunoprecipitation of IRF1 from primary wild-type BMDCs that were stimulated with 25 μg/ml c-di-GMP. Anti-STING was used to detect proteins by Western blotting. f Antibodies detecting phosphoserine and/threonine were used to precipitate IRF1 from HEK293T cells that were co-transfected with HA-STING and HA-IRF1 encoding plasmids. IRF1 and STING were detected using HA antibody. g BMDCs from wild-type and Tmem173−/− (1 × 106 cells per time point) were stimulated with 50 μg/ml of c-di-GMP as indicated. Cells were lysed and analyzed by Western blotting with indicated antibodies (Abs). p-IRF-3 p-STAT1, p-c-Jun, p-p65 were detected first and the membranes stripped and re-probed with anti-IRF-3, anti-STAT1, anti-c-Jun, anti-p65, and anti-β-actin. One of two independent experiments is shown. h Immunoblot analysis of the nuclear recruitment of IRF1 in BMDCs isolated from wild-type or Irf3;Ifnar−/− mice upon STING activation Data are representative of two independent experiments c–h or are from three independent experiments b.
