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. 2020 Mar 2;11:1146. doi: 10.1038/s41467-020-14948-z

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Immunoprecipitation analysis of ubiquitination of endogenous Tyk2, JAK1, STAT1, and STAT2 in HEK293T cells cotransfected with Flag-LUBAC and HA-Ub-K0 (HA-K0, all lysines on Ub are mutated to arginine) using a HA antibody. b Western blot analysis of the JAK-STAT signaling proteins (p-Tyk2, Tyk2, p-JAK1, and JAK1) in HEK293T cells transfected with Flag-LUBAC and then treated with IFNα (1000 IU/ml) as indicated. c Immunoprecipitation analysis of STAT1 ubiquitination in HEK293T cells cotransfected with Myc-STAT1 (WT or its mutants) and HA-K0. d Highly conserved lysine (K) residues (K511 and K652) on STAT1 from different species. e Immunoprecipitation analysis of linear ubiquitination of STAT1 in U3A (Stat1^−/−) cells transfected with Myc-STAT1 (WT), -K511R, -K652R, or -DM (K511/652R). f Immunoprecipitation analysis of the interaction between Flag-HOIP and Myc-STAT1 (WT, K511R, K652R, and DM) in HEK293T cells. g Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T cells cotransfected with Myc-STAT1 (WT or DM) and (or) Flag-LUBAC. h Western blot analysis of pY-STAT1 in U3A cells transfected with Myc-STAT1 (WT or DM) and then treated with IFNα (1000 IU/ml) as indicated. i Dual-luciferase reporter assay of the ISRE activity in HEK293T cells cotransfected with Myc-STAT1 (WT or DM), ISRE luciferase, and Renilla, and then stimulated with IFNα (1000 IU/ml) for 20 h. j RT-qPCR analysis of Ifit1 mRNA in U3A cells transfected with Myc-STAT1 (WT or DM), and then treated with IFNβ (1000 IU/ml) for 4 h. k RT-qPCR analysis of VSV viral RNA in Stat1^−/− MEF cells transfected with Myc-STAT1 (WT or DM), and then treated with mIFNβ (60 IU/ml) for 20 h, followed by infection with VSV (MOI = 0.1) for 24 h. *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed unpaired Student’s t test). Data are shown as mean and s.d. of three biological replicates (i, j, k), or are representative of three independent experiments (a–c, e–h).