Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β promotes activation of STAT1 and STAT2 during HSV-1 skin infection. a , b Expression of Stat1 ( a ) and Stat2 ( b ) mRNAs were examined in wild type and IL-36β KO HSV-1 infected skin (Fig. ). No statistically significant differences were detected. c Quantification of total STAT1 and pSTAT1 in wild type and IL-36β KO HSV-1 infected skin by western blotting and ImageJ analysis (WT, n = 5; KO, n = 5). d STAT2 and pSTAT2 levels in wild type and IL-36β KO HSV-1 infected skin were determined by western blotting and ImageJ analysis (WT, n = 3; KO, n = 4). c , d Representative data from one of three independent experiments involving male mice is shown. Quantitative data are shown as means ± SD. * p < 0.05; ** p < 0.01 (one-way ANOVA). Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Rabbit monoclonal antibodies to phospho-STAT1 (Tyr701, 58D6, Catalog # 9167, RRID: AB_561284, used at 1:800 dilution (Westerns) and 1:50 dilution (immunohistochemistry)), phospho-STAT2 (Tyr690, D3P2P, Catalog # 88410, RRID: AB_2800123, used at 1:800 dilution), STAT1 (D1K9Y, Catalog # 14994, RRID:AB_2799965, used at 1:1000 dilution (Westerns)), STAT2 (D9J7L, Catalog # 72604, RRID:AB_2799824, used at 1:1000 dilution), Gapdh (14C10, Catalog # 2118, RRID: AB_561053, used at 1:2000 dilution), IRF1 (D5E4, Catalog # 8478, RRID:AB_10949108, used at 1:1000 dilution), anti-rabbit IgG HRP-linked (Catalog # 7074, RRID: AB_2099233) or anti-mouse IgG HRP-linked (Catalog # 7076, RRID: AB_330924) antibodies (used at 1:10,000 dilution) were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Infection, Expressing, Western Blot

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β induces STAT1- and STAT2-dependent antiviral immunity in keratinocytes. a Human keratinocytes were pre-treated with medium only or IL-36β before infection with HSV-1 (MOI = 0.01). Levels of HSV-1 ICP4 protein were determined by Western blotting and ImageJ analyses using GAPDH as control. b Mouse primary keratinocytes were pre-treated with medium only or IL-36β, followed by HSV-1 infection (MOI = 0.01), and ICP4 levels examined by western blotting. c Wild type ( + / + ) and IL-36β KO (−/−) mouse primary keratinocytes were infected with 0.01 MOI HSV-1 and ICP4 examined by western blotting. d Wild type and STAT1 −/− primary male mouse keratinocytes were treated with medium only or IL-36β followed by HSV-1 infection (MOI = 0.01) for 24 h. Levels of HSV-1 ICP4 and host Mx1 were examined by western blotting. e Wild type and STAT2 −/− primary male mouse keratinocytes were examined after IL-36β pre-treatment and HSV-1 infection using western blotting. a – e Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group); ** p < 0.01. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Rabbit monoclonal antibodies to phospho-STAT1 (Tyr701, 58D6, Catalog # 9167, RRID: AB_561284, used at 1:800 dilution (Westerns) and 1:50 dilution (immunohistochemistry)), phospho-STAT2 (Tyr690, D3P2P, Catalog # 88410, RRID: AB_2800123, used at 1:800 dilution), STAT1 (D1K9Y, Catalog # 14994, RRID:AB_2799965, used at 1:1000 dilution (Westerns)), STAT2 (D9J7L, Catalog # 72604, RRID:AB_2799824, used at 1:1000 dilution), Gapdh (14C10, Catalog # 2118, RRID: AB_561053, used at 1:2000 dilution), IRF1 (D5E4, Catalog # 8478, RRID:AB_10949108, used at 1:1000 dilution), anti-rabbit IgG HRP-linked (Catalog # 7074, RRID: AB_2099233) or anti-mouse IgG HRP-linked (Catalog # 7076, RRID: AB_330924) antibodies (used at 1:10,000 dilution) were obtained from Cell Signaling Technology.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: IL-36 promotes anti-viral immunity by boosting sensitivity to IFN-α/β in IRF1 dependent and independent manners

doi: 10.1038/s41467-019-12318-y

Figure Lengend Snippet: IL-36β accelerates type I IFN signaling. Medium only or IL-36β treated keratinocytes were further treated with type I IFN (0.01 ng mL −1 ) as indicated. Phosphorylation of STAT1 and STAT2 was examined by western blotting and ImageJ analyses. Quantitative data are shown as means ± SD. * p < 0.05 (one-way ANOVA, n = 2 biologically independent samples per treatment group). a Human keratinocytes were analyzed. b Mouse primary keratinocytes were examined. Each red dot represents a single data point. Source data are provided as a Source Data file

Article Snippet: Rabbit monoclonal antibodies to phospho-STAT1 (Tyr701, 58D6, Catalog # 9167, RRID: AB_561284, used at 1:800 dilution (Westerns) and 1:50 dilution (immunohistochemistry)), phospho-STAT2 (Tyr690, D3P2P, Catalog # 88410, RRID: AB_2800123, used at 1:800 dilution), STAT1 (D1K9Y, Catalog # 14994, RRID:AB_2799965, used at 1:1000 dilution (Westerns)), STAT2 (D9J7L, Catalog # 72604, RRID:AB_2799824, used at 1:1000 dilution), Gapdh (14C10, Catalog # 2118, RRID: AB_561053, used at 1:2000 dilution), IRF1 (D5E4, Catalog # 8478, RRID:AB_10949108, used at 1:1000 dilution), anti-rabbit IgG HRP-linked (Catalog # 7074, RRID: AB_2099233) or anti-mouse IgG HRP-linked (Catalog # 7076, RRID: AB_330924) antibodies (used at 1:10,000 dilution) were obtained from Cell Signaling Technology.

Techniques: Phospho-proteomics, Western Blot

Journal: Oncotarget

Article Title: Comparative proteomics as a tool for identifying specific alterations within interferon response pathways in human glioblastoma multiforme cells

doi: 10.18632/oncotarget.22751

Figure Lengend Snippet: Relative quantities of STAT1 and STAT2 in IFN-resistant A-172 and IFN-sensitive DBTRG-05MG cell lines: real time qPCR ( A ), LC-MS/MS-based LFQ ( B ), western blot ( C ). Whiskers show standard deviations, ±σ. β-Actin equally expressed in the cells under study was used as a reference in western blot analysis.

Article Snippet: Blotted membranes were then incubated with primary Rabbit monoclonal antibodies to Stat2 (D9J7L, Cell Signaling Technology, USA) at 1:1000 dilution, overnight at 4 °C, washed 3 times in PBST for 10 min, and incubated for 1 h at room temperature with secondary HRP-labeled mouse anti-Rabbit antibodies (Santa Cruz Biotechnology) at 1:5000 dilution.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: Nuclear localization of STAT1 and STAT2 is inhibited by HMPV. Human BEAS2b cells were infected with HMPV or mock for 20 h, and treated with IFN for 40 min. After IFN treatment, cells were fixed, stained for immunofluorescence, and images were captured at 40× magnification. The fluorescent intensities of the nuclear and cytosolic STAT1 or STAT2 fluorescence signals was quantified. ( A ) HMPV infection inhibits nuclear translocation of STAT1. ( B ) STAT2 nuclear localization is impaired during HMPV infection. Note reduction of STAT1/2 (red) signal in nuclei of HMPV-infected (green) cells. ** p < 0.01, **** p < 0.0001 compared to Mock + IFN, one-way ANOVA with Tukey post-hoc test. Images are representative of 2 independent experiments. Data in graphs are from two experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Infection, Staining, Immunofluorescence, Fluorescence, Translocation Assay

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: HMPV reduces STAT1 and STAT2 activation in primate cells. ( A ) VeroE6 (primate) cells were infected with HMPV for 24 h, then treated with IFN for 30 min before cells were lysed for Western blotting against total and phosphorylated STAT1 and STAT2. ( B , C ) Fold change of total STAT1 ( B ) and STAT2 ( C ) expression normalized to value of mock-infected cells. ( D ) Fold change of pSTAT1 and pSTAT2 compared to mock-infected cells. ( E ) Relative fold change of pSTAT1 and pSTAT2 (fold change of pSTAT1/2 divided by fold change of STAT1/2). * p < 0.05, ** p < 0.01, one sample t test comparison to normalized mock value of 1. Data are representative of ( A ) or combined from ( B – E ) four independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Activation Assay, Infection, Western Blot, Expressing, Comparison

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: HMPV infection of murine cells leads to upregulation and phosphorylation of STAT1 and STAT2. ( A ) CMT64/61 (murine), left, and NIH3T3 (murine), right, cells were infected with HMPV for 24 h, then treated with IFN for 30 min before cells were lysed for Western blotting against total and phosphorylated STAT1 and STAT2. ( B , C ) Fold change of total STAT1 and STAT2 expression compared to mock-infected wells for CMT64/61 cells. ( D ) Fold change of pSTAT1 and pSTAT2 compared to mock-infected wells in CMT64/61 cells. ( E , F ) For NIH3T3 cells, fold change of STAT1 and STAT2 compared to mock-infected wells. ( G ) Fold change of pSTAT1 and pSTAT2 compared to mock-infected cells in NIH3T3 cells. * p < 0.05, one sample t test comparing samples to normalized mock value of 1. Data are representative of ( A ) or combined from four ( B – D ) or two ( E – G ) independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Infection, Phospho-proteomics, Western Blot, Expressing

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: HMPV does not require STAT1 or STAT2 to inhibit expression and phosphorylation of the other. STAT2-deficient U6A ( A ) and STAT1-deficient U3A ( B ) cells were infected with HMPV for 20 h at an MOI of 1 and 3, treated with IFN for 40 min, and lysed for Western blotting. ( A , B ) Expression and phosphorylation of STAT1 and STAT2 in U6A and U3A cells. ( C ) Fold change of STAT1/pSTAT1 protein levels in U6A cells. ( D ) Fold change of STAT2/pSTAT2 levels in U3A cells. * p < 0.05, ** p < 0.01, one sample t test to normalized mock value of 1. Data are representative of ( A , B ) or combined from ( C , D ) two independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Expressing, Phospho-proteomics, Infection, Western Blot

Journal: Viruses

Article Title: STAT2 Limits Host Species Specificity of Human Metapneumovirus

doi: 10.3390/v12070724

Figure Lengend Snippet: Expression of human STAT2 promotes STAT1 and STAT2 inhibition, while murine STAT2 inhibits STAT degradation. STAT2-deficient U6A cells were transfected with hSTAT2 or mSTAT2, then infected with HMPV. Cells were treated with IFN 16 h after infection for 40 min before cell lysis and protein harvesting for Western blotting. ( A ) STAT1 and STAT2 expression and phosphorylation in U6A cells in the presence of human or murine STAT2. ( B – E ) Quantification of band intensity as a measure of fold change in U6A cells for total STAT1 ( B ), pSTAT1 ( C ), total STAT2 ( D ), and pSTAT2 ( E ). * p < 0.05, *** p < 0.001, one sample t test against normalized mock value of 1. Data are representative of ( A ) or combined from ( B – E ) two independent experiments.

Article Snippet: Antibodies used were STAT1 (CST, D1K9Y) STAT2 (CST, D9J7L), HMPV anti-Fusion protein 54G10 [ ], secondary Alexa Fluor 488-conjugated anti-human and Alexa Fluor 568-conjugated anti-rabbit (Invitrogen).

Techniques: Expressing, Inhibition, Transfection, Infection, Lysis, Western Blot, Phospho-proteomics

Journal: Journal of Innate Immunity

Article Title: Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition

doi: 10.1159/000454926

Figure Lengend Snippet: STAT2 is required for Clr-b induction following type-I interferon (IFN) treatment. a NIH3T3 cells were transfected with a vector encoding the mouse cytomegalovirus M27 immunoevasin or pIRES2-GFP empty vector and then treated with 103 U/mL IFN-α4 for 24 h the next day. Clr-b expression was analyzed by flow cytometry, gated on GFP+ cells. Dotted lines represent the secondary reagent alone; solid lines represent untreated cells; shaded histograms represent IFN-treated cells. b Cells were transfected as in a and then examined by Western blot for STAT2 expression the next day. c ChIP using an anti-STAT2 mAb was performed on NIH3T3 cells treated for 1.5 h with 103 U/mL IFN-α4, using qRT-PCR primers spanning the IRF cluster of the Clec2d promoter or exon 5 (n = 2 experiments). d NIH3T3 cells stably expressing M27 using a modified Dox-inducible piggyBac vector (NIH3T3.M27) were used to perform STAT2 ChIP as in c (n = 2 experiments). **** p < 0.0001.

Article Snippet: Chromatin was precleared with 25 μL Pansorbin (Merck Millipore) at 4°C for 15 min. A 100-μL aliquot of sonicated chromatin was immunoprecipitated with 2 μg STAT2 mAb (D9J7L; Cell Signaling Technology) or normal rabbit IgG (Santa Cruz Biotechnology) at 4°C overnight.

Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Western Blot, Quantitative RT-PCR, Stable Transfection, Modification

Journal: Molecules

Article Title: Dual Inhibition of PB2 and JAK2 for Influenza: A Strategy Combining Antiviral and Host-Directed Immune Modulation

doi: 10.3390/molecules31040696

Figure Lengend Snippet: Effects of PB05 on STAT2 phosphorylation in virus-infected and IFN-β stimulated THP1 cells. ( a ) Western blot analysis of STAT2 phosphorylation in influenza virus-infected THP1 cells treated with PB05, with corresponding quantitative analysis shown on the right. ( b ) Western blot and RT–qPCR analyses of STAT2 activation and downstream gene expression in THP1 cells following direct IFN-β stimulation in the presence of PB05. Statistical significance is indicated as **** p < 0.0001.

Article Snippet: Anti-Stat2 (D9J7L) (#72604) and anti-phospho-Stat2 (Tyr690) (D3P2P) (#88410) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Virus, Infection, Western Blot, Quantitative RT-PCR, Activation Assay, Gene Expression

Journal: Heliyon

Article Title: Innate antiviral responses against Shaan virus infection in HEK293, A549 and MARC-145 cells and limited role of viperin against Shaan virus replication

doi: 10.1016/j.heliyon.2023.e22597

Figure Lengend Snippet: The expression level of IFN-α, -β and -λ1 in Shaan virus-infected HEK293, A549 and MARC-145 cells HEK293, A549, and MARC-145 cells were infected with the Shaan virus (ShaV) at an MOI of 1 or mock. RNA expression levels of IFN-α (A), -β (B) and -λ1 (C) were detected using real time RT-PCR. Data were normalized to GAPDH expression and presented as relative fold expression values for each control cell. P values of <0.05 were considered statistically significant and denoted as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) (n = 3). (D) Western blots of STAT1 and STAT2 proteins in infected cells at the indicated time points. Uncropped images of western blots displayed in .

Article Snippet: The following primary antibodies were used in this study: IRF3 (D83B9), IRF7 (D2A1J), phosphorylated-IRF3 Ser396 (4D4G), STAT1 (D1K9Y), STAT2 (D9J7L), phosphorylated- STAT1 Tyr701 (58D6) and phosphorylated- STAT2 Tyr690 (D3P2P) (Cell signaling Technology), viperin (ab107359), and beta-actin (ab8226) (Abcam).

Techniques: Expressing, Virus, Infection, RNA Expression, Quantitative RT-PCR, Control, Western Blot

Journal: mBio

Article Title: African swine fever virus MGF-360-10L is a novel and crucial virulence factor that mediates ubiquitination and degradation of JAK1 by recruiting the E3 ubiquitin ligase HERC5

doi: 10.1128/mbio.00606-23

Figure Lengend Snippet: MGF-360-10L targets and colocalizes with JAK1. ( A ) HEK293T cells were transfected with Flag-MGF-360-10L expression plasmid or empty vector for 24 h, then treated with IFN-β (1,000 U/mL) for the indicated time. Cell lysates were analyzed by Western blotting with the indicated antibodies. ( B ) PAMs were infected with ASFV-10L-GFP or ASFV-Δ10L (MOI: 0.01) for the indicated time, then treated with IFN-β (0.7 µg/mL) before Western blotting. ( C–E ) HEK293T cells were transfected with Myc-JAK1 (1 µg) ( C ), Myc-JAK2 (1 µg) ( E ), and Flag-MGF-360-10L (0, 0.5, 1, and 2.0 µg) ( D ) for 24 h, and cell lysates were then analyzed by Western blotting with the indicated antibodies. ( F ) PAMs were infected with ASFV-10L-GFP or ASFV-Δ10L, cell lysates were analyzed by Western blotting. ( G and H ) Total RNA in HEK293T ( G ) and PAMs ( H ) were extracted to detect the mRNA levels of JAK1. ( I ) HEK293T cells were transfected with Myc-JAK1, Myc-JAK2, Myc-STAT1, Myc-STAT2, and Flag-MGF-360-10L. Co-immunoprecipitation and Western blotting analyses were performed using the indicated antibodies. ( J ) PAMs were infected with ASFV-10L-GFP for 24 h, and co-immunoprecipitation and Western blotting analyses were performed with the indicated antibodies. ( K ) HEK293T cells were co-transfected with empty vector or Flag-MGF-360-10L and Myc-JAK1 plasmids for 24 h. The subcellular localization of Flag-MGF-360-10L and Myc-JAK1 in the cells were observed using a confocal microscope. Data represent the mean ± SD, n = 3. * P ＜0.05, ** P ＜0.01, *** P ＜0.001. DAPI, 4′, 6′-diamidino-2-phenylindole.

Article Snippet: Polyclonal rabbit anti-JAK1 (29261), phospho-JAK1 (Tyr1034/1035) (3331S), JAK2 (3230), TYK2 (9312S), phospho-TYK2 (Tyr1054/1055) (9321S), STAT1 (14994), phospho-STAT1 (Tyr701) (9167), STAT2 (D9J7L), and phospho-STAT2 (Y690) (AP0284) were purchased from Cell Signaling Technology (USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Infection, Immunoprecipitation, Microscopy

Journal: Frontiers in microbiology

Article Title: Synthetic mimetics assigned a major role to IFNAR2 in type I interferon signaling.

doi: 10.3389/fmicb.2022.947169

Figure Lengend Snippet: FIGURE 1 Native and synthetic type I IFN signaling depends on Jak1 and Tyk2. (A) Schematic overview of native and synthetic type I IFN signaling. Type I IFN signal via IFNAR1 (myc-tagged, lagoon) and IFNAR2 (HA tagged, blue) which are associated with Tyk2 and Jak1, respectively. In the synthetic receptors, the extracellular domains of IFNAR1 and IFNAR2 were replaced by nanobodies directed against GFP (green) and mCherry (red), respectively. Native IFNARs are activated by type I IFNs, while synthetic IFNARs were activated by dimeric GFP/mCherry ligands (GC). (B) STAT1 activation in U4C (Jak1−/−), γ2A (Jak2−/−) and U1A (Tyk2−/−) cells transiently expressing murine IFNAR1 and IFNAR2 treated with 200 U/ml IFNα4, 10 ng/ml Hyper-IL-6 or 10 ng/ml GC for 20 to 30 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT1 and STAT1, Jak1, Jak2, Tyk2, IFNAR1 (myc-tagged), and IFNAR2 (HA-tagged). Western blot data shows one representative experiment out of three. (C) STAT1 activation in U4C (Jak1−/−), γ2A (Jak2−/−), and U1A (Tyk2−/−) cells transiently expressing murine VGIFNAR1 and VCIFNAR2 treated with 200 U/ml IFNα4, 10 ng/ml Hyper-IL-6 or 10 ng/ml GC for 20 to 30 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT1 and STAT1, Jak1, Jak2, Tyk2, IFNAR1 (myc-tagged), and IFNAR2 (HA-tagged). Western blot data shows one representative experiment out of three.

Article Snippet: Phospho-STAT1 [(Tyr701; 58D6), #9167], STAT1 (#9172), phospho-STAT2 [(Tyr690; D3P2P), #88410], STAT2 [(D9J7L), #72604], HA-Tag [(C29F4), #3724], Myc-Tag [(71D10), #2278], Jak1 [(6G4), #3344], Jak2 [(D2E12), #3230], Tyk2 (#9312) antibodies were obtained from Cell Signaling Technology (Frankfurt, Germany).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Frontiers in microbiology

Article Title: Synthetic mimetics assigned a major role to IFNAR2 in type I interferon signaling.

doi: 10.3389/fmicb.2022.947169

Figure Lengend Snippet: FIGURE 2 Comparable pattern and intensities of STAT activation by native and synthetic type I IFN receptors. (A) Whereas native type I IFN signaling depends on IFNAR1/2 heterodimers, the synthetic cytokines can engage different IFNAR1/2 receptor combinations. IFNAR1 (myc-tagged, lagoon) and IFNAR2 (HA-tagged, blue) which are associated with Tyk2 and Jak1, respectively. In the synthetic receptors, the extracellular domains of IFNAR1 and IFNAR2 were replaced by nanobodies directed against GFP (green) and mCherry (red), respectively. Native IFNARs are activated by type I IFNs, synthetic IFNARs were activated by multimeric GFP/mCherry ligands (GC, GG, CC, GCCG). (B) STAT1 and STAT2 activation in HEK293 cells transiently expressing murine IFNAR1 and IFNAR2 (left), murine VGIFNAR1 and VCIFNAR2 (middle) or human VGIFNAR1 and VCIFNAR2 (right) treated with 1,000 U/ml IFNα4, 10 ng/ml Hyper-IL-6 or 100 ng/ml of the synthetic cytokine ligands (GCCG, GG, CC, GC) for 30 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT1, phospho-STAT2 and STAT1, STAT2, IFNAR1 (myc-tagged), and IFNAR2 (HA-tagged). Western blot data shows one representative experiment out of three. (C) STAT1 activation in Ba/F3-gp130 cells after stimulation with 200 U/ml IFNα4, 100 ng/ml IFNβ, 20 ng/ml IFNγ, 20 ng/ml IFNλ2, 10 ng/ml Hyper-IL-6, or 100 ng/ml of the synthetic cytokine ligands GC for 30 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT1 and STAT1. Western blot data shows one representative experiment out of three. (D) Cell surface expression of Ba/F3-gp130 cells transfected with either murine or human VGIFNAR1 and VCIFNAR2. Expression was detected via antibodies detecting myc-tagged IFNAR1 (upper panel) or HA-tagged IFNAR2 (lower panel)

Article Snippet: Phospho-STAT1 [(Tyr701; 58D6), #9167], STAT1 (#9172), phospho-STAT2 [(Tyr690; D3P2P), #88410], STAT2 [(D9J7L), #72604], HA-Tag [(C29F4), #3724], Myc-Tag [(71D10), #2278], Jak1 [(6G4), #3344], Jak2 [(D2E12), #3230], Tyk2 (#9312) antibodies were obtained from Cell Signaling Technology (Frankfurt, Germany).

Techniques: Activation Assay, Expressing, Western Blot, Transfection