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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. 4 — a Immunoprecipitation analysis of the interaction between endogenous JAK1 and STAT1 in HEK293T cells transfected with or without Flag-LUBAC and then treated with IFNα (1000 IU/ml) for 15 min. b Immunoprecipitation analysis of the interaction between endogenous JAK1 and STAT1 in HeLa (left) and HEK293T (right) cells transfected with shHOIP (#1, #2) and then treated with IFNα as a. c Immunoprecipitation analysis of the interaction between HA-IFNAR2 and endogenous STAT1 in HEK293T cells transfected with shHOIP and then treated with IFNα as a. d Immunoprecipitation analysis of the interaction between endogenous IFNAR2 and STAT1 in HEK293T cells treated as b. e Immunoprecipitation analysis of the interaction between HA-IFNAR2 and Myc-STAT1 in HEK293T cells cotransfected with HA-IFNAR2 and Myc-STAT1-WT or Myc-STAT1-K511/652 R (DM) and then treated with IFNα as a. f Immunoprecipitation analysis of the interaction between HA-IFNAR2 and STAT1 (WT or DM) in a plate of U3A cells cotransfected with HA-IFNAR2 (purple rectangle), Flag-STAT1-WT (blue rectangle), and Myc-STAT1-DM (orange rectangle) and then treated with IFNα as a. Double red lines: linear ubiquitination. g In vitro binding assay to analyze the interaction between HA-IFNAR2 and Flag-STAT1 (WT or DM) that were immunoprecipitated from HEK293T cells transfected with either HA-IFNAR2 or Flag-STAT1. h In vitro kinase assay to analyze the phosphorylation effect of HA-JAK1 on Flag-STAT1 (WT or DM). i HEK293T cells were transfected with Myc-IFNAR2 and (or) Flag-STAT1. The Flag-STAT1 proteins binding with Myc-IFNAR2 were immunoprecipitated by Myc antibodies. The supernatant from Myc immunoprecipitation was subjected to further immunoprecipitation using Flag antibodies to pull down those Flag-STAT1 proteins that did not interact with Myc-IFNAR2. Linear ubiquitination was analyzed as indicated. Data are representative of three independent experiments.