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. 2017 Dec 4;214(12):3553–3563. doi: 10.1084/jem.20170180

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© 2017 Yu et al.

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Figure 1. — USP1–UAF1 complex enhances IFN-β production. (A and B) Western blot analysis of UAF1 and USP1 expression in mouse peritoneal macrophages infected with SeV (A) or stimulated with IFN-β (B) for the indicated time periods. (C) Mouse peritoneal macrophages were treated with increasing amounts of ML323 for 4 h and then stimulated with SeV for 18 h. ELISA analyzed IFN-β production. (D) Mouse peritoneal macrophages were treated with 30 µM ML323 for 4 h and then transfected with ISD for 8 h. ELISA analyzed IFN-β production. (E and F) RAW264.7 cells were transfected with IFN-β or NF-κB reporter plasmid together with UAF1 and USP1 expression plasmids or a control plasmid and analyzed for luciferase activity after treatment with LPS for 6 h, poly(I:C) for 6 h, or SeV for 12 h. (G and H) Western blot analysis of UAF1 (G) or USP1 (H) expression in mouse peritoneal macrophages transfected with the indicated siRNA for 36 h. (I and J) ELISA analysis of IFN-β in the supernatants of peritoneal macrophages, as in G and H, stimulated with LPS. (K and L) Mouse peritoneal macrophages were transfected with control siRNA, UAF1 siRNA 3, or USP1 siRNA 1 and then stimulated with LPS for 6 h or poly(I:C) for 6 h or infected with SeV for 18 h. ELISA analysis of IFN-β, TNF-α, and IL-6 in the supernatants. (M) Mouse peritoneal macrophages were transfected with control siRNA, UAF1 siRNA 3, or USP1 siRNA 1 and then transfected with ISD for 8 h. ELISA analysis of IFN-β in the supernatants. (N) Western blot analysis of USP12 or USP46 expression in mouse peritoneal macrophages transfected with the indicated siRNA for 36 h. (O) Mouse peritoneal macrophages were transfected with control siRNA, USP12 siRNA 2, or USP46 siRNA 1 and then infected with SeV for 18 h. ELISA analysis of IFN-β in the supernatants. Data are representative of three experiments (mean ± SD of three samples in C, D, I–M, and O; mean ± SD of six samples in E and F; *, P < 0.05; **, P < 0.01; by Student’s t test). Similar results were obtained in three independent experiments in panels A, B, G, H, and N. Ctrl, control.