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. 2018 Feb 5;9:493. doi: 10.1038/s41467-017-02640-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Paracrine regulation via the JAK/STAT pathway. a, c Protein levels of the pathway components in response to 1 μg/ml poly(I:C), characterised by western blotting and numerical model simulations. In separate experiments WT MEFs were stimulated with poly(I:C) in the presence of α-IFNAR, and Stat1^–/– MEFs were stimulated with poly(I:C), for the indicated times. For each experiment, its control, poly(I:C)-stimulated WT MEFs, is included. Whole-cell extracts were analysed. Representative replicates out of 3 are shown. Trajectories show averages of over 200 independent stochastic simulations; the colour key is located next to protein labels. a Mediators of positive feedback were analysed using antibodies against phosphorylated (active) forms of STAT1 (p-Tyr701), TBK1, IKKα/β and IRF3 (p-Ser396), as well as total STAT1, STAT2, RIG-I, TBK1, and IRF3. (*) = IKK isoform-dependent phosphorylation sites: p-IKKα Ser176/180, p-IKKβ Ser 177/181. c STAT1/2-regulated mediators of double negative feedback were analysed using antibodies against total OAS1A and PKR, as well as for a phosphorylated (active) form of PKR (p-Thr451). b, d mRNA levels of STAT1/2-regulated genes, Stat1, Stat2, Ddx58 (RIG-I), Socs1, Eif2ak2 (PKR) and Oas1a, in response to LPS, IFNβ or poly(I:C). WT MEFs were stimulated with 1000 U/ml IFNβ, 1 μg/ml LPS or 1 μg/ml poly(I:C) in the absence (left) or presence (right) of IFNAR-blocking antibody (α-IFNAR). Stat1^–/– MEFs and RelA^–/– MEFs (right) were also stimulated with 1 μg/ml poly(I:C). Time profiles of relative mRNA levels were obtained with RT-PCR, and then rescaled to absolute numbers using digital PCR measurements (bars represent means ± s.e.m., n ≥ 2, see Supplementary Note for plots of all replicates compared with model simulations)