Fig 2: IFN-I stimulation removes STAT1 linear ubiquitination.a Immunoprecipitation analysis of linear ubiquitination of STAT1 and pY-STAT1 in HEK293T cells treated with IFNα (1000 IU/ml) for 30 min. b Immunoprecipitation analysis of linear ubiquitination of endogenous STAT1 in 2fTGH cells treated with IFNα or IFNβ (1000 IU/ml) as indicated. c Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T cells transfected with shRNAs against OTULIN (shOTULIN, #1 and #2). d Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T cells transfected with Myc-STAT1 (WT or DM) and shOTULIN. e Immunoprecipitation analysis of the interaction between OTULIN and STAT1 in 2fTGH cells treated with IFNα (1000 IU/ml) as indicated. f Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T cells transfected with shOTULIN and then treated with IFNα (1000 IU/ml) as indicated. g, h Immunoprecipitation analysis of the interaction between endogenous IFNAR2 and STAT1 in HEK293T cells transfected with either Flag-OTULIN (g) or shOTULIN (#1, #2) (h), and treated with IFNα (1000 IU/ml) for 15 min. i Western blot analysis of pY-STAT1 in HEK293T cells transfected with shOTULIN and then treated with IFNα (1000 IU/ml) as indicated. j RT-qPCR analysis of the representative ISGs (Ifit1 and Isg54) mRNA in HEK293T cells transfected with Flag-OTULIN and stimulated with IFNα (1000 IU/ml) for 4 h. k RT-qPCR analysis of VSV viral RNA in 2fTGH cells transfected with shOTULIN and then stimulated with IFNα (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 (j, k), or are representative of three independent experiments (a–i).

Fig 3: Tyk2 induces Tyr200/277 phosphorylation of LATS1 that promotes LATS1 activation.(A) IP-IB analysis of endogenous LATS1-IFNAR1 or LATS1-IFNAR2 interaction in mouse primary splenocytes. (B) IP-IB analysis of pan-tyrosine phosphorylation of LATS1 (Pan-pY) in HEK293T treated with IFN-α (1000 IU/ml). (C) IP-IB analysis of pan-pY-LATS1 in HEK293T transfected with shCON or shRNAs against JAK1 or Tyk2 (shJAK1 or shTyk2) and then treated with IFN-α (1000 IU/ml, 30 min). The first lane represents an IgG control for IP. (D) IP-IB analysis of pan-pY-LATS1 in HEK293T cotransfected with Flag-LATS1 and increasing amounts of HA-Tyk2. (E and F) IP-IB analysis of pan-pY-LATS1 in HEK293T cotransfected with HA-Tyk2 and Flag-LATS1 mutants (E) or in Lats1/2−/− MEFs transfected with LATS1 [wild type (WT) or Y200F/Y277F] and HA-Tyk2 (F). (G) Flag-LATS1 proteins were immunoprecipitated from HEK293T cotransfected with Flag-LATS1 and HA-Tyk2 by Flag agarose. The red “y” represents Y200/277. m/z is the mass/charge ratio. The “b”s and “y”s indicate mass spectrometry–identified fragment ions from the N termini (b) and C termini (y) of the peptides after fragmentation. The presented diagrams provided the tandem mass spectra of the identified peptides. (H) In vitro kinase assay using Flag-LATS1 pulled down from HEK293T transfected with Flag-LATS1 (WT or YF/YF) and recombinant Tyk2. (I) IP-IB analysis of LATS1-pan-pY (pY-LATS1) and pS909 (pS-LATS1) in Lats1/2−/− MEFs transfected with LATS1 (WT or Y200F/Y277F) and treated with IFN-α (1000 IU/ml, 30 min). (J) IP-IB analysis of pS909-LATS1 in HEK293T transfected with Flag-LATS1 (WT or its phosphomimetic mutant: Y200E, Y277E, or Y200E/Y277E). Data are representative of three independent experiments (A to F and H to J).

Fig 4: Linear ubiquitination blocks the binding of STAT1 to IFNAR2.a Immunoprecipitation analysis of the interaction between endogenous JAK1 and STAT1 in HEK293T cells transfected with or without Flag-LUBAC and then treated with IFNa (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 IFNa as a. c Immunoprecipitation analysis of the interaction between HA-IFNAR2 and endogenous STAT1 in HEK293T cells transfected with shHOIP and then treated with IFNa 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 IFNa 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 IFNa 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.