FIG 9.

XAF1 stabilizes IRF1 protein by antagonizing CHIP-mediated degradation. (A and B) Immunoprecipitation analysis of the interaction of exogenous XAF1-IRF1 in HEK293T cells in a 10-cm dish transfected with the XAF1 (4 μg) and IRF1 (4 μg) plasmids for 24 h. (C) WT and truncated mutants of IRF1 (2 μg) were cotransfected with Flag-Flag-FGEH (2 μg) or Flag-XAF1 (2 μg) into HEK293T cells seeded in 60-mm dishes for 24 h, and anti-FLAG immunoprecipitates from total cell lysates were subjected to Immunoblot (IB) analysis. (D) Immunofluorescence microscopic colocalization analysis of XAF1 and IRF1. HEK293T cells transfected with the Flag-XAF1 (1 μg) and EV, IRF1-GFP, NLS-GFP, or Mf2-GFP (1 μg) for 24 h were incubated with anti-Flag antibody, and proteins were visualized with secondary antibodies; scale bar, “—” represents 25 μm. (E) Immunoprecipitation analysis of the interaction of exogenous IRF1-CHIP in HEK293T cells in a 10-cm dish transfected with the indicated plasmids for 24 h. (F) The ubiquitination of exogenous IRF1 was analyzed by IP and immunoblot in HEK293T in 60-mm dishes transfected with indicated plasmids for 24 h, followed by MG132 (10 μM) treatment for 6 h. (G) The ubiquitination of endogenous IRF1 was analyzed by IP and immunoblot in the WT and Xaf1^−/− PMs in 10-cm dishes infected with PR8 (MOI 0.1) for 8 h, followed by MG132 (10 μM) treatment for 4 h. (H) Working model of this study. As an identified ISG, XAF1 is regulated by transcription factor IRF1, which in turn interacts with and stabilizes IRF1 by antagonizing CHIP-mediated degradation. Then, IRF1 triggers downstream IRF1-dependent IFN-I and antiviral ISGs production. This positive feedback regulatory loop of XAF1 and IRF1 promotes the host antiviral innate immunity. (A–G) Data are representative results of three independent experiments.