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  • 88
    Thermo Fisher gene exp stat2 hs01013123 m1
    Expression of pro- and anti-inflammatory markers in DM patients. Cytokines driving Th1-, Th2- and Th17 pathways were analyzed in adult and juvenile patients. Expression levels of STAT1 and <t>STAT2</t> were significantly increased in jDM patients, while STAT6 showed a significant increase in aDM muscle biopsies. Overall Th17-associated immune markers were expressed on a very low level. Data are represented as Box-Whiskers blot min-max with mean and standard deviation, level of significance p
    Gene Exp Stat2 Hs01013123 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology stat2
    Truncations in the DENV NS5 protein affect its ability to associate with <t>STAT2,</t> decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.
    Stat2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 875 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc stat2
    STAT1 and <t>STAT2</t> levels are higher in liver stellate cells from senescent subjects. a , b STAT1 ( a ) and STAT2 ( b ) levels in human liver tissues were determined by immunohistochemistry. Staining was more intense in stellate cells in liver tissues from 57-, 60-, and 61-year-old persons than in those from 16-, 23-, and 34-year-old persons. The lower panels show enlargements of the boxed areas in the upper panels. Bars in top, middle, and lower panels: 1 mm, 100 µm, and 10 µm, respectively. Arrowheads indicate stellate cells. c Stellate cells with positively stained nuclei were determined by counting 20 cells in every fifth field of view from each patient sample. Data are expressed as means ± s.e. * p
    Stat2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti stat2
    <t>STAT2</t> recruits USP18 to IFNAR2 (a) IB analysis of WCL and anti-FLAG IP derived from U6A cells 24 hours after co-transfection with plasmids encoding USP18, FLAG-IFNAR2, and increasing amounts of STAT2-Myc expression construct. The relative USP18 binding to IFNAR2 from three independent experiments was quantified and plotted as the ratio of IFNAR2-bound USP18 to total USP18 (right panel). Data are normalized to the maximum binding (lane 4). (b) Recruitment of USP18 and STAT2 to micropatterned IFNAR2 in STAT2-deficient U6A cells as illustrated in the cartoon. U6A cells transfected with HaloTag-mTagBFP-IFNAR2 and mEGFP-USP18 (USP18 –STAT2) (green channel, left image) and U6A cells transfected with HaloTag-mTagBFP-IFNAR2, mEGFP-USP18 and STAT2-TagRFP-T (USP18 +STAT2) (green channel, right image). Representative images of 17 cells analyzed in two independent experiments. (c) Recruitment of USP18 to immobilized IFNAR2 (R2) into micropatterns was quantified in U6A cells by determining the contrast of the fluorescence intensities inside and outside the patterns. For comparison, constitutive binding of STAT2 (positive control) and cytosolic mEGFP (negative control) to micropatterned full length IFNAR2 expressed in U6A cells was quantified. n: number of cells analyzed in two independent experiments. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P
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    88
    Santa Cruz Biotechnology human stat2
    Expression of human <t>STAT2</t> does not influence establishment of the murine IFN-α/β antiviral state but enhances SV5 growth in IFN-α/β-responsive murine cells. (A) Pretreatment of cells with IFN-α/β reduces efficiency of virus replication. Cell lines indicated were incubated with or without murine IFN-β (1,000 U/ml) for 6 h and then infected with 10 PFU of SV5/cell. Supernatants were harvested 48 h later and titrated by plaque assay with CV1 cells. (B) Expression of human STAT2 confers a replication advantage to SV5 in mouse cells. Cell lines were infected with SV5 at the indicated MOI, and supernatants were harvested 48 h later for virus titration. (C) Expression of human STAT2 greatly enhances SV5 replication in mouse cells in the presence of excess exogenous IFN-α/β. The experiment was conducted as for panel B but with 1,000 U of murine IFN-β/ml added at the time the SV5 inoculum was replaced, i.e., after 2 h of adsorption. (D) Replication advantage conferred by human STAT2 allows more-efficient SV5 growth during long-term infection. The methodology was similar to that described for panel B. Infected cells were supplemented with IFN-α/β as indicated (+IFN) from 2 h p.i. until day 4. Cells were passaged to larger plates at the time of harvest on day 4 and grown in the absence of IFN-α/β for the next 7 days, at the end of which time accumulated virus in the supernatant was titrated. Time line insets (all panels), experimental designs.
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    99
    Cell Signaling Technology Inc anti stat2
    Expression of human <t>STAT2</t> does not influence establishment of the murine IFN-α/β antiviral state but enhances SV5 growth in IFN-α/β-responsive murine cells. (A) Pretreatment of cells with IFN-α/β reduces efficiency of virus replication. Cell lines indicated were incubated with or without murine IFN-β (1,000 U/ml) for 6 h and then infected with 10 PFU of SV5/cell. Supernatants were harvested 48 h later and titrated by plaque assay with CV1 cells. (B) Expression of human STAT2 confers a replication advantage to SV5 in mouse cells. Cell lines were infected with SV5 at the indicated MOI, and supernatants were harvested 48 h later for virus titration. (C) Expression of human STAT2 greatly enhances SV5 replication in mouse cells in the presence of excess exogenous IFN-α/β. The experiment was conducted as for panel B but with 1,000 U of murine IFN-β/ml added at the time the SV5 inoculum was replaced, i.e., after 2 h of adsorption. (D) Replication advantage conferred by human STAT2 allows more-efficient SV5 growth during long-term infection. The methodology was similar to that described for panel B. Infected cells were supplemented with IFN-α/β as indicated (+IFN) from 2 h p.i. until day 4. Cells were passaged to larger plates at the time of harvest on day 4 and grown in the absence of IFN-α/β for the next 7 days, at the end of which time accumulated virus in the supernatant was titrated. Time line insets (all panels), experimental designs.
    Anti Stat2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p stat2
    Expression of human <t>STAT2</t> does not influence establishment of the murine IFN-α/β antiviral state but enhances SV5 growth in IFN-α/β-responsive murine cells. (A) Pretreatment of cells with IFN-α/β reduces efficiency of virus replication. Cell lines indicated were incubated with or without murine IFN-β (1,000 U/ml) for 6 h and then infected with 10 PFU of SV5/cell. Supernatants were harvested 48 h later and titrated by plaque assay with CV1 cells. (B) Expression of human STAT2 confers a replication advantage to SV5 in mouse cells. Cell lines were infected with SV5 at the indicated MOI, and supernatants were harvested 48 h later for virus titration. (C) Expression of human STAT2 greatly enhances SV5 replication in mouse cells in the presence of excess exogenous IFN-α/β. The experiment was conducted as for panel B but with 1,000 U of murine IFN-β/ml added at the time the SV5 inoculum was replaced, i.e., after 2 h of adsorption. (D) Replication advantage conferred by human STAT2 allows more-efficient SV5 growth during long-term infection. The methodology was similar to that described for panel B. Infected cells were supplemented with IFN-α/β as indicated (+IFN) from 2 h p.i. until day 4. Cells were passaged to larger plates at the time of harvest on day 4 and grown in the absence of IFN-α/β for the next 7 days, at the end of which time accumulated virus in the supernatant was titrated. Time line insets (all panels), experimental designs.
    P Stat2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc stat2
    Locations of all the ChIP–chip and expression results across chromosome 22q based on Ensembl gene annotations. The gray center track represents the regions of chromosome 22 represented on the microarray, from the centromere ( top left ) to the telomere ( bottom right ). Black bars within the gray track represent the locations of ChIP–chip results from the IFN-γ STAT1 ( top third), IFN-α–STAT1 ( middle ), and <t>IFN-α–STAT2</t> ( bottom third) assays. Open diamonds and black triangles signify STAT-binding sites identified by two or three different ChIP–chip assays, respectively. Expression results from up-regulated (red), down-regulated (green), and unaffected (yellow) genes are located above (sense) and below .
    Stat2, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc phospho stat2
    The correlations between ISGF3 subunits and PBRM1, KDM5C, BAP1 or SEDT2 in the TCGA dataset. Correlations between IRF9, STAT1 or <t>STAT2</t> with indicated genes within the TCGA dataset. N = 533.
    Phospho Stat2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    stat2  (Abcam)
    92
    Abcam stat2
    <t>STAT2</t> was a target of miR-506-3p, and DLX6-AS1 regulated STAT2 expression by targeting miR-506-3p. ( A ) The potential target relationship between STAT2 and miR-506-3p was predicted by the online tool Starbase3.0. ( B and C ) The potential target relationship between STAT2 and miR-506-3p was verified by dual-luciferase reporter assay. ( D and E ) The expression of STAT2 in NB tissues was detected by qRT-PCR and Western blot. ( F and G ) The expression of STAT2 in NB cells was detected by qRT-PCR and Western blot. ( H ) The correlation between STAT2 expression and miR-506-3p expression in NB tissues was analyzed according to Spearman correlation analysis. ( I and J ) The expression of STAT2 in SK-N-SH and LAN-6 cells transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA- STAT2 or miR-506-3p+pcDNA-NC was detected by qRT-PCR and Western blot at 48 h post-transfection. ( K ) The correlation between STAT2 expression and DLX6-AS1 expression in NB tissues was analyzed according to Spearman correlation analysis. ( L and M ) The expression of STAT2 in SK-N-SH and LAN-6 cells transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA-DLX6-AS1 or miR-506-3p+pcDNA-NC was examined by qRT-PCR and Western blot at 48 h post-transfection. * P
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    88
    Santa Cruz Biotechnology rabbit anti stat2
    Phosphorylation of <t>S734-STAT2</t> does not affect the antiproliferative effects of IFN-α. (A) Growth rate of U6A cells stably expressing WT-, S734A- or S734D-STAT2 was measured at 48 h and 72 h by an MTS assay. The inset shows a western blot analysis of STAT2 expression in U6A cells expressing empty vector, WT-, S734A or S734D-STAT2. (B) The same panel of cells was treated with 100 U/ml or 1000 U/ml of IFN-α for 72 h and cell viability determined by MTS assay. Results are presented as the percentage of viable cells in the presence of IFN-α compared against untreated cells. Data shown are from three or four independent experiments and presented as mean±s.e.m.
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    89
    SPSS Inc sigma stat 2 03
    Phosphorylation of <t>S734-STAT2</t> does not affect the antiproliferative effects of IFN-α. (A) Growth rate of U6A cells stably expressing WT-, S734A- or S734D-STAT2 was measured at 48 h and 72 h by an MTS assay. The inset shows a western blot analysis of STAT2 expression in U6A cells expressing empty vector, WT-, S734A or S734D-STAT2. (B) The same panel of cells was treated with 100 U/ml or 1000 U/ml of IFN-α for 72 h and cell viability determined by MTS assay. Results are presented as the percentage of viable cells in the presence of IFN-α compared against untreated cells. Data shown are from three or four independent experiments and presented as mean±s.e.m.
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    98
    Cell Signaling Technology Inc anti phospho stat2
    Phosphorylation of <t>S734-STAT2</t> does not affect the antiproliferative effects of IFN-α. (A) Growth rate of U6A cells stably expressing WT-, S734A- or S734D-STAT2 was measured at 48 h and 72 h by an MTS assay. The inset shows a western blot analysis of STAT2 expression in U6A cells expressing empty vector, WT-, S734A or S734D-STAT2. (B) The same panel of cells was treated with 100 U/ml or 1000 U/ml of IFN-α for 72 h and cell viability determined by MTS assay. Results are presented as the percentage of viable cells in the presence of IFN-α compared against untreated cells. Data shown are from three or four independent experiments and presented as mean±s.e.m.
    Anti Phospho Stat2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson stat2
    Activation of the mitochondrial dependent death pathway requires <t>STAT2.</t> Cells were left untreated or treated with 3,000 U/ml IFN-α for 24 and 48 h. A) Loss of mitochondrial membrane potential was measured by incubation of cells with the fluorescent
    Stat2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    SYSTAT sigma stat 2 03
    Activation of the mitochondrial dependent death pathway requires <t>STAT2.</t> Cells were left untreated or treated with 3,000 U/ml IFN-α for 24 and 48 h. A) Loss of mitochondrial membrane potential was measured by incubation of cells with the fluorescent
    Sigma Stat 2 03, supplied by SYSTAT, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti stat2
    Apigenin enhances IFN-α/β-induced JAK/STAT activation. (a) HepG2-ISRE-Luc2 cells were seeded in 96-well plates (1×10 4 /well) and treated with the various concentrations of apigenin for 2 h, then with 200 U/mL IFN-α or IFN-β for 24 h. (b) HEK293A cells were incubated with indicated concentrations of apigenin for 2 h, then with 2000 U/mL IFN-α for another 1 h. The cell lysates were immunoblotted with phospho-STAT1 (Tyr701), STAT1, <t>phospho-STAT2</t> (Tyr690), or STAT2 antibodies. The quantitative results are shown. (c) HEK293A cells were treated 200 U/mL IFN-α with or without 10 µM bortezomib or the indicated concentrations of apigenin for 24 h. Real-time quantitative reverse transcription-PCR was used to determine the mRNA expression of PKR or 2’,5’-OAS1. The result is presented as induction (n-fold) relative to basal levels in untreated cells. GAPDH was used as an internal control. (*) p
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    Jandel Engineering sigma stat 2 03
    Apigenin enhances IFN-α/β-induced JAK/STAT activation. (a) HepG2-ISRE-Luc2 cells were seeded in 96-well plates (1×10 4 /well) and treated with the various concentrations of apigenin for 2 h, then with 200 U/mL IFN-α or IFN-β for 24 h. (b) HEK293A cells were incubated with indicated concentrations of apigenin for 2 h, then with 2000 U/mL IFN-α for another 1 h. The cell lysates were immunoblotted with phospho-STAT1 (Tyr701), STAT1, <t>phospho-STAT2</t> (Tyr690), or STAT2 antibodies. The quantitative results are shown. (c) HEK293A cells were treated 200 U/mL IFN-α with or without 10 µM bortezomib or the indicated concentrations of apigenin for 24 h. Real-time quantitative reverse transcription-PCR was used to determine the mRNA expression of PKR or 2’,5’-OAS1. The result is presented as induction (n-fold) relative to basal levels in untreated cells. GAPDH was used as an internal control. (*) p
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    91
    The Jackson Laboratory stat2
    Function of the NSs binding-deficient <t>STAT2</t> chimera. The reporter plasmids were cotransfected into HEK293T cells with the expression plasmids for NSs and each STAT2 chimera. Twenty-four h after transfection, cells were treated with IFN-αA/D (500 U/ml) for 18 h or left untreated and then were lysed to measure luciferase activity (upper) or detect protein expression using immunoblotting (lower). The ISRE activity was calculated by dividing RLU in IFN-αA/D-treated cells by the units of cells not treated with IFN-αA/D. The ISRE activity in the absence of NSs was set as 100%. The assays were independently performed in triplicate. The data represent averages with SDs. * * , P
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    SPSS Inc stat 2 03 software
    Function of the NSs binding-deficient <t>STAT2</t> chimera. The reporter plasmids were cotransfected into HEK293T cells with the expression plasmids for NSs and each STAT2 chimera. Twenty-four h after transfection, cells were treated with IFN-αA/D (500 U/ml) for 18 h or left untreated and then were lysed to measure luciferase activity (upper) or detect protein expression using immunoblotting (lower). The ISRE activity was calculated by dividing RLU in IFN-αA/D-treated cells by the units of cells not treated with IFN-αA/D. The ISRE activity in the absence of NSs was set as 100%. The assays were independently performed in triplicate. The data represent averages with SDs. * * , P
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    89
    Santa Cruz Biotechnology phospho stat2
    Overexpression of <t>STAT2</t> and IFNAR2 is sufficient to recapitulate differentiation-dependent changes in neuronal type I IFN-stimulated gene expression. (A) Type I IFN dose-response curves were analyzed in undifferentiated BE(2)-C (closed circles), differentiated BE(2)-C/m (closed squares), and undifferentiated cells transfected with either empty vector (open circles, solid line) or IRF-9-, IFNAR2-, and STAT2-overexpression vectors (open circles, dashed line). Normalized SEAP responses 24 h after stimulation with IFNα-A/D were fit using a variable slope nonlinear regression to calculate EC 50 and Hill slope values. (B) BE(2)-C cells were transfected with the indicated expression plasmid combinations, and EC 50 and Hill slope values were calculated from independently fit type I IFN dose-response curves as noted above in (A). * p
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    Santa Cruz Biotechnology rabbit polyclonal antibody against stat2
    Immunoexpression of JAK/STAT proteins in the epidermis, pemphigus vulgaris, × 400. Immunoexpression of JAK3 in the epidermis: a perilesional skin 14.38 ± 3.61; b skin lesions 18.89 ± 4.67, p > 0.05. Immunoexpression of <t>STAT2</t> in the epidermis: c perilesional skin 15.79 ± 2.06; d skin lesions 17.15 ± 2.81, non-significant. Immunoexpression of STAT4 in the epidermis: e perilesional skin 24.10 ± 3.40; f skin lesions 29.08 ± 4.38, p
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    Image Search Results


    Expression of pro- and anti-inflammatory markers in DM patients. Cytokines driving Th1-, Th2- and Th17 pathways were analyzed in adult and juvenile patients. Expression levels of STAT1 and STAT2 were significantly increased in jDM patients, while STAT6 showed a significant increase in aDM muscle biopsies. Overall Th17-associated immune markers were expressed on a very low level. Data are represented as Box-Whiskers blot min-max with mean and standard deviation, level of significance p

    Journal: Acta Neuropathologica Communications

    Article Title: Differential roles of hypoxia and innate immunity in juvenile and adult dermatomyositis

    doi: 10.1186/s40478-016-0308-5

    Figure Lengend Snippet: Expression of pro- and anti-inflammatory markers in DM patients. Cytokines driving Th1-, Th2- and Th17 pathways were analyzed in adult and juvenile patients. Expression levels of STAT1 and STAT2 were significantly increased in jDM patients, while STAT6 showed a significant increase in aDM muscle biopsies. Overall Th17-associated immune markers were expressed on a very low level. Data are represented as Box-Whiskers blot min-max with mean and standard deviation, level of significance p

    Article Snippet: The qPCR assay identification numbers are as follows: IFNG : Hs00989291_m1; IFNA : Hs00265051_s1; STAT1 : Hs01013989_m1; STAT2 : Hs01013123_m1; STAT3 : Hs00374280_m1; STAT6 : Hs00598625_m1; TNFA : Hs00174128_m1; FBXO32 : Hs01041408_m1; cox2 : Hs00153133_m1; RORG : Hs01076122_m1; IL1B : Hs01555410_m1; IL4 : Hs00929862_m1; IL5 : Hs01548712_g1; IL6 : Hs00985639_m1; IL12B : Hs01011518_m1; IL13 : Hs99999038_m1; IL17 : Hs00174383_m1; IL21 : Hs00222327_m1; IL27 : Hs00377366_m1; ISG15 : Hs01921425_s1; OAS3 : Hs00196324_m1, MX1 : Hs00895608_m1; RIPK : Hs00169407_m1; MDA5 : Hs01070332_m1; CCL17 : Hs00171074_m1; MRC1 (CD206): Hs00267207_m1; TGFB : Hs00998133_m1; VEGFA : Hs00900055_m1; HIF1A : Hs00153153_m1; MIF : Hs00236988_g1; PGK : Hs99999906_m1.

    Techniques: Expressing, Standard Deviation

    Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.

    Journal: Journal of Virology

    Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

    doi: 10.1128/JVI.02188-08

    Figure Lengend Snippet: Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.

    Article Snippet: Although the binding of DENV NS5 to STAT2 was sufficient to prevent IFN signaling, the degradation of STAT2 might result in an additional advantage, since once STAT2 bound to NS5 is degraded, the NS5 molecule that was previously bound to STAT2 might now become free to perform other functions, such as RNA transcription or additional degradation of other STAT2 molecules.

    Techniques: Transfection, Construct, FACS, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay

    Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.

    Journal: Journal of Virology

    Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

    doi: 10.1128/JVI.02188-08

    Figure Lengend Snippet: Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.

    Article Snippet: Although the binding of DENV NS5 to STAT2 was sufficient to prevent IFN signaling, the degradation of STAT2 might result in an additional advantage, since once STAT2 bound to NS5 is degraded, the NS5 molecule that was previously bound to STAT2 might now become free to perform other functions, such as RNA transcription or additional degradation of other STAT2 molecules.

    Techniques: Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, FACS

    DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.

    Journal: Journal of Virology

    Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

    doi: 10.1128/JVI.02188-08

    Figure Lengend Snippet: DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.

    Article Snippet: Although the binding of DENV NS5 to STAT2 was sufficient to prevent IFN signaling, the degradation of STAT2 might result in an additional advantage, since once STAT2 bound to NS5 is degraded, the NS5 molecule that was previously bound to STAT2 might now become free to perform other functions, such as RNA transcription or additional degradation of other STAT2 molecules.

    Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

    Reduced levels of STAT2 in cells expressing the nonstructural region of the DENV polyprotein. (A) Vero cells were infected for 24 h with DENV at an MOI of 10 and subsequently lysed and examined by Western blotting. (B) U6A cells stably expressing STAT2-GFP were infected with DEN2 at an MOI of 40 for 24 h prior to fixation. Cells were then probed with antibody against NS5 and stained for DNA (DAPI [4′,6′-diamidino-2-phenylindole]). (C) U6A-STAT2-GFP cells were infected with DEN2 at an MOI of 40 and measured for a loss of GFP by live microscopy at the given time points. White arrows indicate cells in the infected samples at 9 h p.i. that have lost the GFP signal. (D) Vero cells were infected with DENV at an MOI of 10, subsequently lysed at the given time points, and examined by Western blotting. (E) Vero cells stably expressing a DEN1 replicon (NS1-5) were lysed and examined by Western blotting. (F) wtVero cells or Vero cells stably expressing a DEN1 replicon were treated with the indicated amounts of type I IFN (IFN-α/β) for 24 h. Cells were subsequently challenged with NDV-GFP and assayed via fluorescence microscopy for GFP expression at 14 h p.i. (G) Vero cells or Vero cells stably expressing a DEN1 replicon were treated with 1,000 units of type I (IFN-α/β) or type II (IFN-γ) IFN for 24 h. Cells were subsequently challenged with NDV-GFP and assayed via fluorescence microscopy for GFP expression at 14 h p.i. (H) 293T cells were cotransfected with plasmids expressing NS1-5-HA and GFP or NS1-4b-HA and GFP. Twenty-four hours posttransfection, cells were sorted by FACS for GFP-positive cells and subsequently lysed and examined via Western blotting. Densitometry analysis of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with the value of 1 in the case of NS5 levels being indicative of no detection (background levels).

    Journal: Journal of Virology

    Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

    doi: 10.1128/JVI.02188-08

    Figure Lengend Snippet: Reduced levels of STAT2 in cells expressing the nonstructural region of the DENV polyprotein. (A) Vero cells were infected for 24 h with DENV at an MOI of 10 and subsequently lysed and examined by Western blotting. (B) U6A cells stably expressing STAT2-GFP were infected with DEN2 at an MOI of 40 for 24 h prior to fixation. Cells were then probed with antibody against NS5 and stained for DNA (DAPI [4′,6′-diamidino-2-phenylindole]). (C) U6A-STAT2-GFP cells were infected with DEN2 at an MOI of 40 and measured for a loss of GFP by live microscopy at the given time points. White arrows indicate cells in the infected samples at 9 h p.i. that have lost the GFP signal. (D) Vero cells were infected with DENV at an MOI of 10, subsequently lysed at the given time points, and examined by Western blotting. (E) Vero cells stably expressing a DEN1 replicon (NS1-5) were lysed and examined by Western blotting. (F) wtVero cells or Vero cells stably expressing a DEN1 replicon were treated with the indicated amounts of type I IFN (IFN-α/β) for 24 h. Cells were subsequently challenged with NDV-GFP and assayed via fluorescence microscopy for GFP expression at 14 h p.i. (G) Vero cells or Vero cells stably expressing a DEN1 replicon were treated with 1,000 units of type I (IFN-α/β) or type II (IFN-γ) IFN for 24 h. Cells were subsequently challenged with NDV-GFP and assayed via fluorescence microscopy for GFP expression at 14 h p.i. (H) 293T cells were cotransfected with plasmids expressing NS1-5-HA and GFP or NS1-4b-HA and GFP. Twenty-four hours posttransfection, cells were sorted by FACS for GFP-positive cells and subsequently lysed and examined via Western blotting. Densitometry analysis of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with the value of 1 in the case of NS5 levels being indicative of no detection (background levels).

    Article Snippet: Although the binding of DENV NS5 to STAT2 was sufficient to prevent IFN signaling, the degradation of STAT2 might result in an additional advantage, since once STAT2 bound to NS5 is degraded, the NS5 molecule that was previously bound to STAT2 might now become free to perform other functions, such as RNA transcription or additional degradation of other STAT2 molecules.

    Techniques: Expressing, Infection, Western Blot, Stable Transfection, Staining, Microscopy, Fluorescence, FACS

    Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.

    Journal: Journal of Virology

    Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

    doi: 10.1128/JVI.02188-08

    Figure Lengend Snippet: Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.

    Article Snippet: Although the binding of DENV NS5 to STAT2 was sufficient to prevent IFN signaling, the degradation of STAT2 might result in an additional advantage, since once STAT2 bound to NS5 is degraded, the NS5 molecule that was previously bound to STAT2 might now become free to perform other functions, such as RNA transcription or additional degradation of other STAT2 molecules.

    Techniques: Expressing, Transfection, Construct, FACS, Western Blot, Mutagenesis, Labeling

    Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.

    Journal: Journal of Virology

    Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

    doi: 10.1128/JVI.02188-08

    Figure Lengend Snippet: Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.

    Article Snippet: Although the binding of DENV NS5 to STAT2 was sufficient to prevent IFN signaling, the degradation of STAT2 might result in an additional advantage, since once STAT2 bound to NS5 is degraded, the NS5 molecule that was previously bound to STAT2 might now become free to perform other functions, such as RNA transcription or additional degradation of other STAT2 molecules.

    Techniques: Transfection, Construct, FACS, Polyacrylamide Gel Electrophoresis, Western Blot

    STAT1 and STAT2 levels are higher in liver stellate cells from senescent subjects. a , b STAT1 ( a ) and STAT2 ( b ) levels in human liver tissues were determined by immunohistochemistry. Staining was more intense in stellate cells in liver tissues from 57-, 60-, and 61-year-old persons than in those from 16-, 23-, and 34-year-old persons. The lower panels show enlargements of the boxed areas in the upper panels. Bars in top, middle, and lower panels: 1 mm, 100 µm, and 10 µm, respectively. Arrowheads indicate stellate cells. c Stellate cells with positively stained nuclei were determined by counting 20 cells in every fifth field of view from each patient sample. Data are expressed as means ± s.e. * p

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: ISGF3 with reduced phosphorylation is associated with constitutive expression of interferon-induced genes in aging cells

    doi: 10.1038/s41514-018-0030-6

    Figure Lengend Snippet: STAT1 and STAT2 levels are higher in liver stellate cells from senescent subjects. a , b STAT1 ( a ) and STAT2 ( b ) levels in human liver tissues were determined by immunohistochemistry. Staining was more intense in stellate cells in liver tissues from 57-, 60-, and 61-year-old persons than in those from 16-, 23-, and 34-year-old persons. The lower panels show enlargements of the boxed areas in the upper panels. Bars in top, middle, and lower panels: 1 mm, 100 µm, and 10 µm, respectively. Arrowheads indicate stellate cells. c Stellate cells with positively stained nuclei were determined by counting 20 cells in every fifth field of view from each patient sample. Data are expressed as means ± s.e. * p

    Article Snippet: The antibodies used were as follows: anti-STAT1 (#14994, CST, 1:100), -STAT2 (#72604, CST, 1:100), -IRF9 (#76684, CST, 1:100), and -8-OHdG (#MOG-20P; JaICA, Shizuoka, Japan).

    Techniques: Immunohistochemistry, Staining

    Unphosphorylated STAT1 and STAT2 levels are increased in fibroblasts from a patient with Werner syndrome. a SA-β-gal staining in fibroblasts from a patient with Werner syndrome at passage 8 (p8) and in NHDFs at passage 13 (p13). SA-β-gal staining was more intense in fibroblasts at passage 8 compared with NHDFs at passage 13. Representative results from three independent experiments are shown. Bars in upper and lower panels, 50 and 10 µm. Positive cells were determined by counting 20 cells in every fifth field of view from three experiments in each group. Data are expressed as the means ± s.e. * p

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: ISGF3 with reduced phosphorylation is associated with constitutive expression of interferon-induced genes in aging cells

    doi: 10.1038/s41514-018-0030-6

    Figure Lengend Snippet: Unphosphorylated STAT1 and STAT2 levels are increased in fibroblasts from a patient with Werner syndrome. a SA-β-gal staining in fibroblasts from a patient with Werner syndrome at passage 8 (p8) and in NHDFs at passage 13 (p13). SA-β-gal staining was more intense in fibroblasts at passage 8 compared with NHDFs at passage 13. Representative results from three independent experiments are shown. Bars in upper and lower panels, 50 and 10 µm. Positive cells were determined by counting 20 cells in every fifth field of view from three experiments in each group. Data are expressed as the means ± s.e. * p

    Article Snippet: The antibodies used were as follows: anti-STAT1 (#14994, CST, 1:100), -STAT2 (#72604, CST, 1:100), -IRF9 (#76684, CST, 1:100), and -8-OHdG (#MOG-20P; JaICA, Shizuoka, Japan).

    Techniques: Staining

    Unphosphorylated STAT levels are increased in the nucleus of senescent NHDFs. a STAT1 and STAT2 protein levels were increased in NHDFs at passage 23 (p23), compared with passage 5 (p5). Representative results from three independent experiments are shown. b Phosphorylated STAT1 and STAT2 levels were not increased in NHDFs at passage 23 (p23). STAT1, STAT2, IRF9, and Mx1 protein levels were increased; the STAT3 protein level was unchanged; and the SIRT1 protein level was decreased in the nucleus of NHDFs. Cell lysates from Huh7 cells treated with IFNα for 12 h were used as controls. Representative results from three independent experiments are shown. nc negative control, ns nonspecific bands. c Unphosphorylated STAT1 and STAT2 protein levels were increased in the nucleus of NHDFs at passage 23 (p23) compared with passage 5 (p5). Cytoplasmic (C) and nuclear (N) fractions were extracted from NHDFs and subjected to western blotting. HSP70 and histone H3 proteins were used as controls for cytoplasmic and nuclear proteins, respectively. Whole-cell lysates from Huh7 cells treated with IFNα for 12 h were used as controls. Representative results from three independent experiments are shown. nc negative control. d The levels of all phosphorylated forms of STAT1 and STAT2 were determined by phos-tag gel analyses. The levels of unphosphorylated STAT1 and STAT2 proteins were increased in NHDFs at passage 23 (p23) compared with passage 5 (p5). Lysates of Huh7 cells treated with IFNα for 12 h were used as controls. Representative results from three independent experiments are shown. nc negative control. The numbers below the panel indicate the band intensities for the phosphorylated forms (upper) and nonphosphorylated forms (lower)

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: ISGF3 with reduced phosphorylation is associated with constitutive expression of interferon-induced genes in aging cells

    doi: 10.1038/s41514-018-0030-6

    Figure Lengend Snippet: Unphosphorylated STAT levels are increased in the nucleus of senescent NHDFs. a STAT1 and STAT2 protein levels were increased in NHDFs at passage 23 (p23), compared with passage 5 (p5). Representative results from three independent experiments are shown. b Phosphorylated STAT1 and STAT2 levels were not increased in NHDFs at passage 23 (p23). STAT1, STAT2, IRF9, and Mx1 protein levels were increased; the STAT3 protein level was unchanged; and the SIRT1 protein level was decreased in the nucleus of NHDFs. Cell lysates from Huh7 cells treated with IFNα for 12 h were used as controls. Representative results from three independent experiments are shown. nc negative control, ns nonspecific bands. c Unphosphorylated STAT1 and STAT2 protein levels were increased in the nucleus of NHDFs at passage 23 (p23) compared with passage 5 (p5). Cytoplasmic (C) and nuclear (N) fractions were extracted from NHDFs and subjected to western blotting. HSP70 and histone H3 proteins were used as controls for cytoplasmic and nuclear proteins, respectively. Whole-cell lysates from Huh7 cells treated with IFNα for 12 h were used as controls. Representative results from three independent experiments are shown. nc negative control. d The levels of all phosphorylated forms of STAT1 and STAT2 were determined by phos-tag gel analyses. The levels of unphosphorylated STAT1 and STAT2 proteins were increased in NHDFs at passage 23 (p23) compared with passage 5 (p5). Lysates of Huh7 cells treated with IFNα for 12 h were used as controls. Representative results from three independent experiments are shown. nc negative control. The numbers below the panel indicate the band intensities for the phosphorylated forms (upper) and nonphosphorylated forms (lower)

    Article Snippet: The antibodies used were as follows: anti-STAT1 (#14994, CST, 1:100), -STAT2 (#72604, CST, 1:100), -IRF9 (#76684, CST, 1:100), and -8-OHdG (#MOG-20P; JaICA, Shizuoka, Japan).

    Techniques: Negative Control, Western Blot

    ZIKV antagonizes type I IFN signaling. (A, B) A549 cells were infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOIs of 0.1 and 1. At 48hpi, cells were pulse treated with 1000 IU/mL of recombinant human IFNβ for 30 minutes and whole-cell lysates were collected for western blot analysis of phospho-STAT1 (Tyr701), phospho-STAT2 (Tyr689), STAT1, STAT2, and GAPDH. Representative blots are shown from one of two independent experiments. Quantitation is shown below the representative blots, where intensity values are represented as the ratio of pSTAT:total STAT protein. (C) moDCs were infected with PR-2015 (MOI 10) and STAT1 and STAT2 signaling was assessed as in A and B. Data is representative of three donors from two independent experiments. Quantitation is shown to the right of the representative blots, where intensity values are represented as the ratio of pSTAT:total STAT protein.

    Journal: PLoS Pathogens

    Article Title: Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1006164

    Figure Lengend Snippet: ZIKV antagonizes type I IFN signaling. (A, B) A549 cells were infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOIs of 0.1 and 1. At 48hpi, cells were pulse treated with 1000 IU/mL of recombinant human IFNβ for 30 minutes and whole-cell lysates were collected for western blot analysis of phospho-STAT1 (Tyr701), phospho-STAT2 (Tyr689), STAT1, STAT2, and GAPDH. Representative blots are shown from one of two independent experiments. Quantitation is shown below the representative blots, where intensity values are represented as the ratio of pSTAT:total STAT protein. (C) moDCs were infected with PR-2015 (MOI 10) and STAT1 and STAT2 signaling was assessed as in A and B. Data is representative of three donors from two independent experiments. Quantitation is shown to the right of the representative blots, where intensity values are represented as the ratio of pSTAT:total STAT protein.

    Article Snippet: Western blot analysis was performed to detect STAT1 phosphotyrosine residue 701 (Cell Signaling), total STAT1 (Cell Signaling), STAT2 phosphotyrosine residue 689 (Upstate, EMD Milipore), total STAT2 (Cell Signaling), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling).

    Techniques: Infection, Recombinant, Western Blot, Quantitation Assay

    STAT2 recruits USP18 to IFNAR2 (a) IB analysis of WCL and anti-FLAG IP derived from U6A cells 24 hours after co-transfection with plasmids encoding USP18, FLAG-IFNAR2, and increasing amounts of STAT2-Myc expression construct. The relative USP18 binding to IFNAR2 from three independent experiments was quantified and plotted as the ratio of IFNAR2-bound USP18 to total USP18 (right panel). Data are normalized to the maximum binding (lane 4). (b) Recruitment of USP18 and STAT2 to micropatterned IFNAR2 in STAT2-deficient U6A cells as illustrated in the cartoon. U6A cells transfected with HaloTag-mTagBFP-IFNAR2 and mEGFP-USP18 (USP18 –STAT2) (green channel, left image) and U6A cells transfected with HaloTag-mTagBFP-IFNAR2, mEGFP-USP18 and STAT2-TagRFP-T (USP18 +STAT2) (green channel, right image). Representative images of 17 cells analyzed in two independent experiments. (c) Recruitment of USP18 to immobilized IFNAR2 (R2) into micropatterns was quantified in U6A cells by determining the contrast of the fluorescence intensities inside and outside the patterns. For comparison, constitutive binding of STAT2 (positive control) and cytosolic mEGFP (negative control) to micropatterned full length IFNAR2 expressed in U6A cells was quantified. n: number of cells analyzed in two independent experiments. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: STAT2 recruits USP18 to IFNAR2 (a) IB analysis of WCL and anti-FLAG IP derived from U6A cells 24 hours after co-transfection with plasmids encoding USP18, FLAG-IFNAR2, and increasing amounts of STAT2-Myc expression construct. The relative USP18 binding to IFNAR2 from three independent experiments was quantified and plotted as the ratio of IFNAR2-bound USP18 to total USP18 (right panel). Data are normalized to the maximum binding (lane 4). (b) Recruitment of USP18 and STAT2 to micropatterned IFNAR2 in STAT2-deficient U6A cells as illustrated in the cartoon. U6A cells transfected with HaloTag-mTagBFP-IFNAR2 and mEGFP-USP18 (USP18 –STAT2) (green channel, left image) and U6A cells transfected with HaloTag-mTagBFP-IFNAR2, mEGFP-USP18 and STAT2-TagRFP-T (USP18 +STAT2) (green channel, right image). Representative images of 17 cells analyzed in two independent experiments. (c) Recruitment of USP18 to immobilized IFNAR2 (R2) into micropatterns was quantified in U6A cells by determining the contrast of the fluorescence intensities inside and outside the patterns. For comparison, constitutive binding of STAT2 (positive control) and cytosolic mEGFP (negative control) to micropatterned full length IFNAR2 expressed in U6A cells was quantified. n: number of cells analyzed in two independent experiments. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P

    Article Snippet: Reagents and antibodies Antibodies were commercially purchased as followed; anti-phospho-JAK1 (Tyr1022/1023) (Cell Signaling), anti-JAK1 (Cell Signaling), anti-phospho-STAT1 (Tyr 701) (Cell Signaling), anti- STAT1 (Cell Signaling), anti-STAT2 (Santa Cruz), anti-green fluorescent protein (anti-GFP), and anti-tubulin (Sigma).

    Techniques: Derivative Assay, Cotransfection, Expressing, Construct, Binding Assay, Transfection, Fluorescence, Positive Control, Negative Control

    Inhibiting negative feedback regulation of USP18 by targeting its interaction with STAT2 (a) 293T cells were co-transfected with plasmids encoding STAT1-FLAG, FLAG-USP18 and either STAT2 CC/DB-Myc or the mutant STAT2 CC/DB L227A R409A K415A-Myc (3A). Following treatment with IFNα (1000 U/ml) for 15 minutes as indicated cell lysates were collected and immunoblotted with indicated antibodies. The ratio of p-STAT1/total STAT1 was quantified by LI-COR Odyssey system. (b) Cells indicated in Fig. 7a were treated with IFNα (1000 U/ml) for 12 hours and then expression of GBP1 was analyzed by RT q-PCR. Data represents mean ± S.D. for two independent experiments. (c) THP-1 cells transduced with either MIP control (−) or MIP-STAT2 CC/DB 3A (3A) were treated with IFNα (1000 U/ml) for 48 hours and then Annexin V positive cells were analyzed by flow cytometry. Data represents mean ± S.E.M. for three independently generated stable cell lines. ** P

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: Inhibiting negative feedback regulation of USP18 by targeting its interaction with STAT2 (a) 293T cells were co-transfected with plasmids encoding STAT1-FLAG, FLAG-USP18 and either STAT2 CC/DB-Myc or the mutant STAT2 CC/DB L227A R409A K415A-Myc (3A). Following treatment with IFNα (1000 U/ml) for 15 minutes as indicated cell lysates were collected and immunoblotted with indicated antibodies. The ratio of p-STAT1/total STAT1 was quantified by LI-COR Odyssey system. (b) Cells indicated in Fig. 7a were treated with IFNα (1000 U/ml) for 12 hours and then expression of GBP1 was analyzed by RT q-PCR. Data represents mean ± S.D. for two independent experiments. (c) THP-1 cells transduced with either MIP control (−) or MIP-STAT2 CC/DB 3A (3A) were treated with IFNα (1000 U/ml) for 48 hours and then Annexin V positive cells were analyzed by flow cytometry. Data represents mean ± S.E.M. for three independently generated stable cell lines. ** P

    Article Snippet: Reagents and antibodies Antibodies were commercially purchased as followed; anti-phospho-JAK1 (Tyr1022/1023) (Cell Signaling), anti-JAK1 (Cell Signaling), anti-phospho-STAT1 (Tyr 701) (Cell Signaling), anti- STAT1 (Cell Signaling), anti-STAT2 (Santa Cruz), anti-green fluorescent protein (anti-GFP), and anti-tubulin (Sigma).

    Techniques: Transfection, Mutagenesis, Expressing, Polymerase Chain Reaction, Transduction, Flow Cytometry, Cytometry, Generated, Stable Transfection

    STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA lentivirus. After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA lentivirus. After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.

    Article Snippet: Reagents and antibodies Antibodies were commercially purchased as followed; anti-phospho-JAK1 (Tyr1022/1023) (Cell Signaling), anti-JAK1 (Cell Signaling), anti-phospho-STAT1 (Tyr 701) (Cell Signaling), anti- STAT1 (Cell Signaling), anti-STAT2 (Santa Cruz), anti-green fluorescent protein (anti-GFP), and anti-tubulin (Sigma).

    Techniques: Inhibition, Expressing, Infection, Western Blot, shRNA, Selection

    Both the coiled-coil (CC) and DNA binding (DB) domains of STAT2 are involved in the interaction with USP18 (a) A schematic drawing of STAT2 domain structure and the associated deletion mutants used in this study. The ability of a given deletion mutant to interact with USP18 (+ or − binding) is indicated to the right. (b–c) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding FLAG-USP18 and either the full-length STAT2-Myc (WT) or the indicated deletion mutants. (d) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding GFP-USP18 and either STAT2-FLAG or the indicated deletion mutants. Asterisks are used to indicate non-specific bands. HC = Heavy Chain, LC = Light Chain.

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: Both the coiled-coil (CC) and DNA binding (DB) domains of STAT2 are involved in the interaction with USP18 (a) A schematic drawing of STAT2 domain structure and the associated deletion mutants used in this study. The ability of a given deletion mutant to interact with USP18 (+ or − binding) is indicated to the right. (b–c) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding FLAG-USP18 and either the full-length STAT2-Myc (WT) or the indicated deletion mutants. (d) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding GFP-USP18 and either STAT2-FLAG or the indicated deletion mutants. Asterisks are used to indicate non-specific bands. HC = Heavy Chain, LC = Light Chain.

    Article Snippet: Reagents and antibodies Antibodies were commercially purchased as followed; anti-phospho-JAK1 (Tyr1022/1023) (Cell Signaling), anti-JAK1 (Cell Signaling), anti-phospho-STAT1 (Tyr 701) (Cell Signaling), anti- STAT1 (Cell Signaling), anti-STAT2 (Santa Cruz), anti-green fluorescent protein (anti-GFP), and anti-tubulin (Sigma).

    Techniques: Binding Assay, Mutagenesis, Derivative Assay, Cotransfection

    The coiled-coil (CC) and DNA binding (DB) domains of STAT2 are important for USP18-mediated inhibition of Type I IFN signaling (a) U6A cells, stably transduced with control (−) or C-terminally Myc-tagged STAT2 (STAT2-Myc), were infected with MIP control (−) or MIP-USP18 (+) retrovirus. With or without IFNα (1000 U/ml) treatment for 15 minutes cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (b–d) U6A cells, stably transduced to express either full-length STAT2 (STAT2-Myc), or the indicated STAT2 deletion mutant (b, STAT2ΔCC-Myc; c, STAT2ΔDB-Myc; d, STAT2ΔCC/DB-Myc) with or without IFNα (1000 U/ml) treatment for 15 minutes cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (e) Histograms showing the surface expression of IFNAR1 and IFNAR2 following infection with indicated constructs in U6A cell lines.

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: The coiled-coil (CC) and DNA binding (DB) domains of STAT2 are important for USP18-mediated inhibition of Type I IFN signaling (a) U6A cells, stably transduced with control (−) or C-terminally Myc-tagged STAT2 (STAT2-Myc), were infected with MIP control (−) or MIP-USP18 (+) retrovirus. With or without IFNα (1000 U/ml) treatment for 15 minutes cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (b–d) U6A cells, stably transduced to express either full-length STAT2 (STAT2-Myc), or the indicated STAT2 deletion mutant (b, STAT2ΔCC-Myc; c, STAT2ΔDB-Myc; d, STAT2ΔCC/DB-Myc) with or without IFNα (1000 U/ml) treatment for 15 minutes cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (e) Histograms showing the surface expression of IFNAR1 and IFNAR2 following infection with indicated constructs in U6A cell lines.

    Article Snippet: Reagents and antibodies Antibodies were commercially purchased as followed; anti-phospho-JAK1 (Tyr1022/1023) (Cell Signaling), anti-JAK1 (Cell Signaling), anti-phospho-STAT1 (Tyr 701) (Cell Signaling), anti- STAT1 (Cell Signaling), anti-STAT2 (Santa Cruz), anti-green fluorescent protein (anti-GFP), and anti-tubulin (Sigma).

    Techniques: Binding Assay, Inhibition, Stable Transfection, Transduction, Infection, Western Blot, Mutagenesis, Expressing, Construct

    STAT2-USP18 interaction regulates ternary complex assembly of the Type I IFN receptor (a) Binding of DY647 IFNα2 M148A bound to the IFNAR at the surface of U6A cells expressing STAT2-TagRFP-T (top-left), USP18-mEGFP (top-right) or both (bottom-left). The images are superimpositions of single molecule localizations from 100 consecutive frames. Scale bars: 10 μm. Representative images of 13–18 cells analyzed for each condition. (b) Comparison of the density of DY647 IFNα2 M148A bound to cell surface IFNAR of U6A cells expressing STAT2, USP18 or both proteins. Furthermore, protein with only STAT2 CC domain or with only CC and DB domains of STAT2 were also used in the assay. As a control, localizations of DY647 IFNα2 M148A on the surface of U6A cells transfected with mEGFP were quantified. Data were acquired in two independent experiments, n indicates number of cells analyzed for each condition. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: STAT2-USP18 interaction regulates ternary complex assembly of the Type I IFN receptor (a) Binding of DY647 IFNα2 M148A bound to the IFNAR at the surface of U6A cells expressing STAT2-TagRFP-T (top-left), USP18-mEGFP (top-right) or both (bottom-left). The images are superimpositions of single molecule localizations from 100 consecutive frames. Scale bars: 10 μm. Representative images of 13–18 cells analyzed for each condition. (b) Comparison of the density of DY647 IFNα2 M148A bound to cell surface IFNAR of U6A cells expressing STAT2, USP18 or both proteins. Furthermore, protein with only STAT2 CC domain or with only CC and DB domains of STAT2 were also used in the assay. As a control, localizations of DY647 IFNα2 M148A on the surface of U6A cells transfected with mEGFP were quantified. Data were acquired in two independent experiments, n indicates number of cells analyzed for each condition. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P

    Article Snippet: Reagents and antibodies Antibodies were commercially purchased as followed; anti-phospho-JAK1 (Tyr1022/1023) (Cell Signaling), anti-JAK1 (Cell Signaling), anti-phospho-STAT1 (Tyr 701) (Cell Signaling), anti- STAT1 (Cell Signaling), anti-STAT2 (Santa Cruz), anti-green fluorescent protein (anti-GFP), and anti-tubulin (Sigma).

    Techniques: Binding Assay, Expressing, Transfection

    USP18 interacts with STAT2 (a) Yeast two-hybrid analysis of the direct interaction between USP18 and STAT2. (a),(b) Overnight cultures from strains pJ69-4a/pJ69-4alpha carrying two-hybrid plasmids with the indicated coding sequences were spotted onto selective media for plasmid maintenance (-Leu, -Tryp = Ctrl for growth control) and indicators for interactions (-His, in addition to -Leu, -Tryp). (b) Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-FLAG immunoprecipitates (IP) derived from 293T cells 24 hours after co-transfection with plasmids encoding GFP, GFP-USP18, and STAT2-FLAG. The number for left side of column shows molecular weight (kDa). (c) GST pull-down assay to demonstrate that the STAT2 directly associate with USP18. (d) Immobilization of STAT2 for probing direct interaction with USP18 as depicted in the cartoon. U5A cells transfected with mEGFP-USP18 (green channel) and STAT2 intracellular fused to a transmembrane domain (TMD) and extracellular fused to mTagBFP as well as HaloTag (blue channel). Scale bars: 10 μm. Representative image of 36 cells analyzed. Intensity profiles of all channels within the yellow ROI depicted in the merged image (right panel). Quantitative analysis of the recruitment of mEGFP-USP18 to micropatterned STAT2 as determined by the contrast of the fluorescence intensities inside and outside the patterns. As negative controls, U5A cells were transfected with mEGFP-USP18 and HaloTag-mTagBFP-TMD as well as mEGFP and HaloTag-mTagBFP-TMD-STAT2. n: number of cells analyzed obtained from two independent experiments. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P

    Journal: Nature structural & molecular biology

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    doi: 10.1038/nsmb.3378

    Figure Lengend Snippet: USP18 interacts with STAT2 (a) Yeast two-hybrid analysis of the direct interaction between USP18 and STAT2. (a),(b) Overnight cultures from strains pJ69-4a/pJ69-4alpha carrying two-hybrid plasmids with the indicated coding sequences were spotted onto selective media for plasmid maintenance (-Leu, -Tryp = Ctrl for growth control) and indicators for interactions (-His, in addition to -Leu, -Tryp). (b) Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-FLAG immunoprecipitates (IP) derived from 293T cells 24 hours after co-transfection with plasmids encoding GFP, GFP-USP18, and STAT2-FLAG. The number for left side of column shows molecular weight (kDa). (c) GST pull-down assay to demonstrate that the STAT2 directly associate with USP18. (d) Immobilization of STAT2 for probing direct interaction with USP18 as depicted in the cartoon. U5A cells transfected with mEGFP-USP18 (green channel) and STAT2 intracellular fused to a transmembrane domain (TMD) and extracellular fused to mTagBFP as well as HaloTag (blue channel). Scale bars: 10 μm. Representative image of 36 cells analyzed. Intensity profiles of all channels within the yellow ROI depicted in the merged image (right panel). Quantitative analysis of the recruitment of mEGFP-USP18 to micropatterned STAT2 as determined by the contrast of the fluorescence intensities inside and outside the patterns. As negative controls, U5A cells were transfected with mEGFP-USP18 and HaloTag-mTagBFP-TMD as well as mEGFP and HaloTag-mTagBFP-TMD-STAT2. n: number of cells analyzed obtained from two independent experiments. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P

    Article Snippet: Reagents and antibodies Antibodies were commercially purchased as followed; anti-phospho-JAK1 (Tyr1022/1023) (Cell Signaling), anti-JAK1 (Cell Signaling), anti-phospho-STAT1 (Tyr 701) (Cell Signaling), anti- STAT1 (Cell Signaling), anti-STAT2 (Santa Cruz), anti-green fluorescent protein (anti-GFP), and anti-tubulin (Sigma).

    Techniques: Plasmid Preparation, Derivative Assay, Cotransfection, Molecular Weight, Pull Down Assay, Transfection, Fluorescence

    The PYTK motif within the SH2 domain of STAT1, STAT2 and STAT3. (A) The conserved motif PYTK (boxed) is conserved in STAT1, STAT2 and STAT3 (B) Domains of human STAT2. The position of PYTK motif located within the SH2 domain is marked. C-C indicates coil-coil

    Journal:

    Article Title: A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis

    doi: 10.1091/mbc.E06-09-0843

    Figure Lengend Snippet: The PYTK motif within the SH2 domain of STAT1, STAT2 and STAT3. (A) The conserved motif PYTK (boxed) is conserved in STAT1, STAT2 and STAT3 (B) Domains of human STAT2. The position of PYTK motif located within the SH2 domain is marked. C-C indicates coil-coil

    Article Snippet: Nuclear extracts were subjected to immunoprecipitation with anti-STAT2 antibody (C20, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by the addition of protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) for another 2 h at 4°C.

    Techniques:

    STAT2 Y631F augments ISGF3-mediated gene expression. Total RNA was isolated and qRT-PCR was performed in triplicate wells using specific primers to the indicated genes. 18S primers were included as an internal control to normalize for equal amount of

    Journal:

    Article Title: A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis

    doi: 10.1091/mbc.E06-09-0843

    Figure Lengend Snippet: STAT2 Y631F augments ISGF3-mediated gene expression. Total RNA was isolated and qRT-PCR was performed in triplicate wells using specific primers to the indicated genes. 18S primers were included as an internal control to normalize for equal amount of

    Article Snippet: Nuclear extracts were subjected to immunoprecipitation with anti-STAT2 antibody (C20, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by the addition of protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) for another 2 h at 4°C.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    STAT2 Y631F inhibits STAT1 tyrosine dephosphorylation. (A) Cells were stimulated with IFN-α (1000 U/ml) for 30 min followed by pulse chase with 500 ng/ml staurosporine (STS) for the indicated times. Whole-cell extracts were prepared and immunoblots

    Journal:

    Article Title: A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis

    doi: 10.1091/mbc.E06-09-0843

    Figure Lengend Snippet: STAT2 Y631F inhibits STAT1 tyrosine dephosphorylation. (A) Cells were stimulated with IFN-α (1000 U/ml) for 30 min followed by pulse chase with 500 ng/ml staurosporine (STS) for the indicated times. Whole-cell extracts were prepared and immunoblots

    Article Snippet: Nuclear extracts were subjected to immunoprecipitation with anti-STAT2 antibody (C20, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by the addition of protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) for another 2 h at 4°C.

    Techniques: De-Phosphorylation Assay, Pulse Chase, Western Blot

    STAT2 Y631F prolongs STAT1 activation. Nuclear extracts were prepared from cells incubated with or without IFN-α for the indicated times. (A) Nuclear extracts were resolved by SDS-PAGE and immunoblot analysis was performed with antibodies against

    Journal:

    Article Title: A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis

    doi: 10.1091/mbc.E06-09-0843

    Figure Lengend Snippet: STAT2 Y631F prolongs STAT1 activation. Nuclear extracts were prepared from cells incubated with or without IFN-α for the indicated times. (A) Nuclear extracts were resolved by SDS-PAGE and immunoblot analysis was performed with antibodies against

    Article Snippet: Nuclear extracts were subjected to immunoprecipitation with anti-STAT2 antibody (C20, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by the addition of protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) for another 2 h at 4°C.

    Techniques: Activation Assay, Incubation, SDS Page

    STAT2 Y631F stimulates the induction of apoptosis by type I IFNs. (A) U6A (STAT2−/−) cells or U6A reconstituted with wild-type STAT2 or STAT2 Y631F were stimulated with either 500 or 3000 U/ml IFN-α and growth inhibition was measured

    Journal:

    Article Title: A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis

    doi: 10.1091/mbc.E06-09-0843

    Figure Lengend Snippet: STAT2 Y631F stimulates the induction of apoptosis by type I IFNs. (A) U6A (STAT2−/−) cells or U6A reconstituted with wild-type STAT2 or STAT2 Y631F were stimulated with either 500 or 3000 U/ml IFN-α and growth inhibition was measured

    Article Snippet: Nuclear extracts were subjected to immunoprecipitation with anti-STAT2 antibody (C20, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by the addition of protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) for another 2 h at 4°C.

    Techniques: Inhibition

    STAT2 Y631F-mediated IFN-α–induced Apoptosis is JAK/STAT Pathway Dependent

    Journal:

    Article Title: A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis

    doi: 10.1091/mbc.E06-09-0843

    Figure Lengend Snippet: STAT2 Y631F-mediated IFN-α–induced Apoptosis is JAK/STAT Pathway Dependent

    Article Snippet: Nuclear extracts were subjected to immunoprecipitation with anti-STAT2 antibody (C20, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by the addition of protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) for another 2 h at 4°C.

    Techniques:

    Time-course kinetics of IFN-β expression and activation of the JAK–STAT signaling pathway in A2MC2-infected MARC-145 cells. (A). Time-course kinetics of IFN-β expression. The cells were infected with A2MC2 at 1 TCID 50 per cell and harvested at 2, 4, 6, 8, 10, 12, and 24 hpi for real-time RT-PCR detection of IFN-β transcript. Relative fold of induction in comparison with uninfected cells are shown. Error bars represent variation between repeated experiments. (B) Viral RNA levels detected by real-time RT-PCR. Relative fold of viral RNA in comparison with that detected at 2 hpi are shown. (C) Activation of the JAK-STAT signaling pathway. The cells were infected with A2MC2 at an MOI of 1 TCID 50 per cell and harvested at 0, 9, 16, and 24 hpi for Western blot analysis of phosphorylated STAT1 (STAT1-Y701) and STAT2 (STAT2-Y690), whole STAT2, and ISG56.

    Journal: Virology

    Article Title: Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate

    doi: 10.1016/j.virol.2012.05.015

    Figure Lengend Snippet: Time-course kinetics of IFN-β expression and activation of the JAK–STAT signaling pathway in A2MC2-infected MARC-145 cells. (A). Time-course kinetics of IFN-β expression. The cells were infected with A2MC2 at 1 TCID 50 per cell and harvested at 2, 4, 6, 8, 10, 12, and 24 hpi for real-time RT-PCR detection of IFN-β transcript. Relative fold of induction in comparison with uninfected cells are shown. Error bars represent variation between repeated experiments. (B) Viral RNA levels detected by real-time RT-PCR. Relative fold of viral RNA in comparison with that detected at 2 hpi are shown. (C) Activation of the JAK-STAT signaling pathway. The cells were infected with A2MC2 at an MOI of 1 TCID 50 per cell and harvested at 0, 9, 16, and 24 hpi for Western blot analysis of phosphorylated STAT1 (STAT1-Y701) and STAT2 (STAT2-Y690), whole STAT2, and ISG56.

    Article Snippet: Briefly, separated proteins from SDS-PAGE were transferred onto a nitrocellulose membrane and probed with antibodies against STAT2 (Santa Cruz Biotechnology, Santa Cruz, CA), β-tubulin (Sigma), phospho-STAT2 (STAT2-Y690) (Santa Cruz Biotechnology), phospho-STAT1 (STAT1-Y701) (Millipore, Billerica, MA), and ISG56 (Thermo Fisher Scientific, Rockford, IL).

    Techniques: Expressing, Activation Assay, Infection, Quantitative RT-PCR, Western Blot

    A2MC2 induces expression of IFN-stimulated genes in primary porcine pulmonary alveolar macrophages (PAMs). (A) STAT2 and IFI56 detected by Western blotting. PAMs were infected with PRRSV VR-2385, A2MC2, and MLV, and at 12 hpi, treated with or without IFN-α. The cells were harvested at 20 hpi for Western blotting. Cell lysate samples from uninfected PAMs with or without IFN treatment were included as controls. (B) IFN bioassay in CRL2843 cells. Supernatant from A2MC2-infected PAMs was diluted and added to the CRL2843 cells 12 h before NDV-GFP inoculation. The cells were observed 24 h after NDV-GFP inoculation. Treatment of the cells with swine IFN-α at a final concentration of 1000 U/ml was included as a positive control. (C) A2MC2 induces elevation of STAT2 in PAM cells from different piglets. PAMs from three piglets were inoculated with A2MC2 at 0.05 TCID 50 per cell, respectively, and incubated for 20 h. Cell lysate samples from IFN-α-treated PAM cells were included as positive controls. Cell lysate samples from non-infected cells were included as negative controls in the Western blot analyses.

    Journal: Virology

    Article Title: Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate

    doi: 10.1016/j.virol.2012.05.015

    Figure Lengend Snippet: A2MC2 induces expression of IFN-stimulated genes in primary porcine pulmonary alveolar macrophages (PAMs). (A) STAT2 and IFI56 detected by Western blotting. PAMs were infected with PRRSV VR-2385, A2MC2, and MLV, and at 12 hpi, treated with or without IFN-α. The cells were harvested at 20 hpi for Western blotting. Cell lysate samples from uninfected PAMs with or without IFN treatment were included as controls. (B) IFN bioassay in CRL2843 cells. Supernatant from A2MC2-infected PAMs was diluted and added to the CRL2843 cells 12 h before NDV-GFP inoculation. The cells were observed 24 h after NDV-GFP inoculation. Treatment of the cells with swine IFN-α at a final concentration of 1000 U/ml was included as a positive control. (C) A2MC2 induces elevation of STAT2 in PAM cells from different piglets. PAMs from three piglets were inoculated with A2MC2 at 0.05 TCID 50 per cell, respectively, and incubated for 20 h. Cell lysate samples from IFN-α-treated PAM cells were included as positive controls. Cell lysate samples from non-infected cells were included as negative controls in the Western blot analyses.

    Article Snippet: Briefly, separated proteins from SDS-PAGE were transferred onto a nitrocellulose membrane and probed with antibodies against STAT2 (Santa Cruz Biotechnology, Santa Cruz, CA), β-tubulin (Sigma), phospho-STAT2 (STAT2-Y690) (Santa Cruz Biotechnology), phospho-STAT1 (STAT1-Y701) (Millipore, Billerica, MA), and ISG56 (Thermo Fisher Scientific, Rockford, IL).

    Techniques: Expressing, Western Blot, Infection, Concentration Assay, Positive Control, Incubation

    Detection of antiviral activity in cell culture supernatant from A2MC2-infected MARC-145 cells. (A) Inhibition of NDV-GFP replication in Vero cells. Vero cells were treated with dilutions of cell culture supernatant of A2MC2-infected MARC-145 cells. The Vero cells were inoculated with NDV-GFP 12 h after the treatment, and observed under fluorescence microscopy at 24 h post-infection. Treatment of the cells with IFN-α at a final concentration of 1000 U/ml was included as a positive control. (B) Elevation of STAT2 and ISG56 proteins in Vero cells after treatment with the supernatant from A2MC2-infected MARC-145 cells detected by Western blot analysis. Vero cells treated with IFN-α and mock-treated were included as positive and negative controls, respectively. Blotting with β-tubulin antibody was done to normalize protein loading. (C) Inhibition of A2MC2 replication in MARC-145 cells by PRRSV-specific peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) 5UP1. A scrambled control PPMO CP1 was included as a negative control. An indirect immunofluorescence assay with PRRSV N-specific monoclonal antibody was conducted. The bottom panel of images shows the nuclear DNA stained with 4′,6-19 diamidino-2-phenylindole (DAPI). (D) Detection of PRRSV proteins in whole cell lysate of A2MC2-infected cells (A2 lane) by Western blotting with pig antiserum. Cell lysate samples from PRRSV VR-2385-infected (VR lane) or MLV-infected cells were included as positive controls. Molecular weight markers are illustrated on the left.

    Journal: Virology

    Article Title: Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate

    doi: 10.1016/j.virol.2012.05.015

    Figure Lengend Snippet: Detection of antiviral activity in cell culture supernatant from A2MC2-infected MARC-145 cells. (A) Inhibition of NDV-GFP replication in Vero cells. Vero cells were treated with dilutions of cell culture supernatant of A2MC2-infected MARC-145 cells. The Vero cells were inoculated with NDV-GFP 12 h after the treatment, and observed under fluorescence microscopy at 24 h post-infection. Treatment of the cells with IFN-α at a final concentration of 1000 U/ml was included as a positive control. (B) Elevation of STAT2 and ISG56 proteins in Vero cells after treatment with the supernatant from A2MC2-infected MARC-145 cells detected by Western blot analysis. Vero cells treated with IFN-α and mock-treated were included as positive and negative controls, respectively. Blotting with β-tubulin antibody was done to normalize protein loading. (C) Inhibition of A2MC2 replication in MARC-145 cells by PRRSV-specific peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) 5UP1. A scrambled control PPMO CP1 was included as a negative control. An indirect immunofluorescence assay with PRRSV N-specific monoclonal antibody was conducted. The bottom panel of images shows the nuclear DNA stained with 4′,6-19 diamidino-2-phenylindole (DAPI). (D) Detection of PRRSV proteins in whole cell lysate of A2MC2-infected cells (A2 lane) by Western blotting with pig antiserum. Cell lysate samples from PRRSV VR-2385-infected (VR lane) or MLV-infected cells were included as positive controls. Molecular weight markers are illustrated on the left.

    Article Snippet: Briefly, separated proteins from SDS-PAGE were transferred onto a nitrocellulose membrane and probed with antibodies against STAT2 (Santa Cruz Biotechnology, Santa Cruz, CA), β-tubulin (Sigma), phospho-STAT2 (STAT2-Y690) (Santa Cruz Biotechnology), phospho-STAT1 (STAT1-Y701) (Millipore, Billerica, MA), and ISG56 (Thermo Fisher Scientific, Rockford, IL).

    Techniques: Activity Assay, Cell Culture, Infection, Inhibition, Fluorescence, Microscopy, Concentration Assay, Positive Control, Western Blot, Negative Control, Immunofluorescence, Staining, Molecular Weight

    A2MC2 replication induces elevated expression of IFN-stimulated genes in MARC-145 cells. (A) Elevation of STAT2 and ISG56 detected by Western blotting. The cells were infected with A2MC2 or UV-inactivated virus at 1 TCID 50 per cell, followed by treatment with PPMO 5UP1 to inhibit A2MC2 replication, and at 24 hpi, treated with or without IFN-α. Cell lysate from uninfected cells was included as a control. (B) Elevation of IFN-β, ISG15 and ISG56 expression detected by real-time PCR. Treatment of the cells with IFN-α was included as a control. Relative induction folds in comparison with mock-treated cells are shown. Error bars represent variation between repeated experiments. Significant differences between A2MC2-infected cells and the uninfected cells are denoted by “*”, which indicates a P -value of

    Journal: Virology

    Article Title: Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate

    doi: 10.1016/j.virol.2012.05.015

    Figure Lengend Snippet: A2MC2 replication induces elevated expression of IFN-stimulated genes in MARC-145 cells. (A) Elevation of STAT2 and ISG56 detected by Western blotting. The cells were infected with A2MC2 or UV-inactivated virus at 1 TCID 50 per cell, followed by treatment with PPMO 5UP1 to inhibit A2MC2 replication, and at 24 hpi, treated with or without IFN-α. Cell lysate from uninfected cells was included as a control. (B) Elevation of IFN-β, ISG15 and ISG56 expression detected by real-time PCR. Treatment of the cells with IFN-α was included as a control. Relative induction folds in comparison with mock-treated cells are shown. Error bars represent variation between repeated experiments. Significant differences between A2MC2-infected cells and the uninfected cells are denoted by “*”, which indicates a P -value of

    Article Snippet: Briefly, separated proteins from SDS-PAGE were transferred onto a nitrocellulose membrane and probed with antibodies against STAT2 (Santa Cruz Biotechnology, Santa Cruz, CA), β-tubulin (Sigma), phospho-STAT2 (STAT2-Y690) (Santa Cruz Biotechnology), phospho-STAT1 (STAT1-Y701) (Millipore, Billerica, MA), and ISG56 (Thermo Fisher Scientific, Rockford, IL).

    Techniques: Expressing, Western Blot, Infection, Real-time Polymerase Chain Reaction

    STAT 1Y701F inhibits late STAT 1‐independent, STAT 2/ IRF 9‐dependent ISG expression in response to IFN β Expression of Stat1 and Stat2 genes. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F , and Stat1 −/− mice were treated with 5 ng/ml of IFN γ ( IFN g) for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in uninduced wild‐type cells. Expression of type I IFN ‐induced genes. BMDM s of wild‐type ( WT ), Stat1 Y701F , Stat1 −/− , Stat2 −/− , and Irf9 −/− mice were treated with 250 IU /ml of IFN β for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in uninduced wild‐type cells. Data information: The bars in (A) and (B) represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ) and asterisks denote level of statistical significance (ns, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001); the P ‐values were calculated using paired ratio t ‐test.

    Journal: EMBO Reports

    Article Title: Response to interferons and antibacterial innate immunity in the absence of tyrosine‐phosphorylated STAT1

    doi: 10.15252/embr.201540726

    Figure Lengend Snippet: STAT 1Y701F inhibits late STAT 1‐independent, STAT 2/ IRF 9‐dependent ISG expression in response to IFN β Expression of Stat1 and Stat2 genes. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F , and Stat1 −/− mice were treated with 5 ng/ml of IFN γ ( IFN g) for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in uninduced wild‐type cells. Expression of type I IFN ‐induced genes. BMDM s of wild‐type ( WT ), Stat1 Y701F , Stat1 −/− , Stat2 −/− , and Irf9 −/− mice were treated with 250 IU /ml of IFN β for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in uninduced wild‐type cells. Data information: The bars in (A) and (B) represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ) and asterisks denote level of statistical significance (ns, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001); the P ‐values were calculated using paired ratio t ‐test.

    Article Snippet: ChIP was performed using STAT1 (clone E‐23, Santa Cruz) or STAT2 antibody (clone C‐20, Santa Cruz).

    Techniques: Expressing, Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction

    Interferon signaling in Listeria monocytogenes ‐infected Stat1 Y701F bone marrow‐derived macrophages Western blot analysis of STAT 1 tyrosine phosphorylation. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice were infected with L. monocytogenes ( LO 28, MOI 10) for 5 or 6 h. Whole‐cell extracts were collected and tested in Western blot for levels of total STAT 1 and phosphorylation of STAT 1 on tyrosine 701 (Y701). The blots are representative of more than three independent experiments. Impact of Stat1 Y701F mutation, or of deletion of ISGF 3 subunits on the expression of the Ifnb gene. BMDM s of wild‐type ( WT ), Stat1 Y701F , Stat1 −/− , Stat2 −/− , and Irf9 −/− mice were infected with L. monocytogenes ( LO 28, MOI 10) for 4, 8, 12, 24, or 48 h. Levels of Ifnb gene expression were determined by qPCR . Bars represent mean values of three independent experiments. Error bars represent standard error of the mean ( SEM ).

    Journal: EMBO Reports

    Article Title: Response to interferons and antibacterial innate immunity in the absence of tyrosine‐phosphorylated STAT1

    doi: 10.15252/embr.201540726

    Figure Lengend Snippet: Interferon signaling in Listeria monocytogenes ‐infected Stat1 Y701F bone marrow‐derived macrophages Western blot analysis of STAT 1 tyrosine phosphorylation. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice were infected with L. monocytogenes ( LO 28, MOI 10) for 5 or 6 h. Whole‐cell extracts were collected and tested in Western blot for levels of total STAT 1 and phosphorylation of STAT 1 on tyrosine 701 (Y701). The blots are representative of more than three independent experiments. Impact of Stat1 Y701F mutation, or of deletion of ISGF 3 subunits on the expression of the Ifnb gene. BMDM s of wild‐type ( WT ), Stat1 Y701F , Stat1 −/− , Stat2 −/− , and Irf9 −/− mice were infected with L. monocytogenes ( LO 28, MOI 10) for 4, 8, 12, 24, or 48 h. Levels of Ifnb gene expression were determined by qPCR . Bars represent mean values of three independent experiments. Error bars represent standard error of the mean ( SEM ).

    Article Snippet: ChIP was performed using STAT1 (clone E‐23, Santa Cruz) or STAT2 antibody (clone C‐20, Santa Cruz).

    Techniques: Infection, Derivative Assay, Western Blot, Mouse Assay, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction

    STAT 1Y701F mutant reduces IFN β‐stimulated nuclear translocation of STAT 2 Analysis of STAT 2 nuclear translocation by immunofluorescence. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice were seeded on cover slips and stimulated with 250 IU /ml of IFN β for 30 min or 24 h. The cells were fixed and stained for STAT 2‐specific antibody followed by Alexa Fluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm. Quantitative evaluation of STAT 2 nuclear translocation. The intensity of STAT 2‐dependent immunofluorescence over DNA staining ( DAPI ) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation ( SD ) and asterisks denote the level of statistical significance (*** P ≤ 0.001); P ‐value was calculated using unpaired t ‐test.

    Journal: EMBO Reports

    Article Title: Response to interferons and antibacterial innate immunity in the absence of tyrosine‐phosphorylated STAT1

    doi: 10.15252/embr.201540726

    Figure Lengend Snippet: STAT 1Y701F mutant reduces IFN β‐stimulated nuclear translocation of STAT 2 Analysis of STAT 2 nuclear translocation by immunofluorescence. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice were seeded on cover slips and stimulated with 250 IU /ml of IFN β for 30 min or 24 h. The cells were fixed and stained for STAT 2‐specific antibody followed by Alexa Fluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm. Quantitative evaluation of STAT 2 nuclear translocation. The intensity of STAT 2‐dependent immunofluorescence over DNA staining ( DAPI ) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation ( SD ) and asterisks denote the level of statistical significance (*** P ≤ 0.001); P ‐value was calculated using unpaired t ‐test.

    Article Snippet: ChIP was performed using STAT1 (clone E‐23, Santa Cruz) or STAT2 antibody (clone C‐20, Santa Cruz).

    Techniques: Mutagenesis, Translocation Assay, Immunofluorescence, Derivative Assay, Mouse Assay, Staining, Software, Standard Deviation

    Deletion of ISGF 3 subunits or Stat1 Y701F mutation is without effect on the expression or IFN β‐stimulated phosphorylation of STAT 3 and STAT 5 Western blot analysis of STAT 3 and STAT 5 tyrosine phosphorylation. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice were treated with 250 IU /ml of IFN β for 0.5, 6, 12, or 24 h. Whole‐cell extracts were collected and tested in Western blot for total STAT 3 and STAT 5 amounts and for their tyrosine phosphorylation at Y705 and Y694, respectively. The blots are representative of more than three independent experiments.

    Journal: EMBO Reports

    Article Title: Response to interferons and antibacterial innate immunity in the absence of tyrosine‐phosphorylated STAT1

    doi: 10.15252/embr.201540726

    Figure Lengend Snippet: Deletion of ISGF 3 subunits or Stat1 Y701F mutation is without effect on the expression or IFN β‐stimulated phosphorylation of STAT 3 and STAT 5 Western blot analysis of STAT 3 and STAT 5 tyrosine phosphorylation. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice were treated with 250 IU /ml of IFN β for 0.5, 6, 12, or 24 h. Whole‐cell extracts were collected and tested in Western blot for total STAT 3 and STAT 5 amounts and for their tyrosine phosphorylation at Y705 and Y694, respectively. The blots are representative of more than three independent experiments.

    Article Snippet: ChIP was performed using STAT1 (clone E‐23, Santa Cruz) or STAT2 antibody (clone C‐20, Santa Cruz).

    Techniques: Mutagenesis, Expressing, Western Blot, Derivative Assay, Mouse Assay

    STAT 1 expression and interferon signaling in Stat1 Y701F mice Effect of Stat1 Y701F homozygosity on STAT 1 expression in mouse cells and organs. Spleens, livers, or bone marrow‐derived macrophages ( BMDM s) were isolated from wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice. Whole‐cell extracts were collected and tested for levels of total STAT1 in Western blot. The blots are representative of more than three independent experiments. Effect of Stat1 Y701F homozygosity on STAT 1 phosphorylation at Y701. BMDM s were isolated from wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice and stimulated for 30 min with 250 IU /ml of IFN β or 5 ng/ml of IFN γ. Whole‐cell extracts were collected and tested for levels of STAT 1 phosphorylation on Y701 in Western blot. The blots are representative of more than three independent experiments. Effect of Stat1 Y701F homozygosity on the expression of IFN β‐induced genes. BMDM s of wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice were treated with 5 ng/ml of IFN γ for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ); asterisks denote the level of statistical significance (ns, P > 0.05); and the P ‐values were calculated using paired ratio t ‐test. Effect of Stat1 Y701F homozygosity on the expression of type I IFN ‐induced genes ( ISG s). BMDM s were isolated from wild‐type ( WT ), Stat1 Y701F , Stat1 −/− , Stat2 −/− , and Irf9 −/− mice treated with 250 IU /ml of IFN β for 4, 8, 12, 24, or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ); asterisks denote the level of statistical significance (* P ≤ 0.05; ** P ≤ 0.01); and the P ‐values were calculated using paired ratio t ‐test. STAT 1 and STAT 2 phosphorylation in Stat1 −/− , Stat1 Y701F , Stat2 −/− , and Irf9 −/− macrophages. BMDM s were isolated from wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice and treated with 250 IU /ml of IFN β for 30 min or 6, 12, or 24 h. Whole‐cell extracts were collected and tested in Western blot for levels of phosphorylation of STAT 1 on Y701 and of STAT 2 on Y689. The same cell extracts were tested for total levels of STAT 1 and STAT 2. The blots are representative of more than three independent experiments.

    Journal: EMBO Reports

    Article Title: Response to interferons and antibacterial innate immunity in the absence of tyrosine‐phosphorylated STAT1

    doi: 10.15252/embr.201540726

    Figure Lengend Snippet: STAT 1 expression and interferon signaling in Stat1 Y701F mice Effect of Stat1 Y701F homozygosity on STAT 1 expression in mouse cells and organs. Spleens, livers, or bone marrow‐derived macrophages ( BMDM s) were isolated from wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice. Whole‐cell extracts were collected and tested for levels of total STAT1 in Western blot. The blots are representative of more than three independent experiments. Effect of Stat1 Y701F homozygosity on STAT 1 phosphorylation at Y701. BMDM s were isolated from wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice and stimulated for 30 min with 250 IU /ml of IFN β or 5 ng/ml of IFN γ. Whole‐cell extracts were collected and tested for levels of STAT 1 phosphorylation on Y701 in Western blot. The blots are representative of more than three independent experiments. Effect of Stat1 Y701F homozygosity on the expression of IFN β‐induced genes. BMDM s of wild‐type ( WT ), Stat1 Y701F ( YF ), and Stat1 −/− (S1) mice were treated with 5 ng/ml of IFN γ for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ); asterisks denote the level of statistical significance (ns, P > 0.05); and the P ‐values were calculated using paired ratio t ‐test. Effect of Stat1 Y701F homozygosity on the expression of type I IFN ‐induced genes ( ISG s). BMDM s were isolated from wild‐type ( WT ), Stat1 Y701F , Stat1 −/− , Stat2 −/− , and Irf9 −/− mice treated with 250 IU /ml of IFN β for 4, 8, 12, 24, or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ); asterisks denote the level of statistical significance (* P ≤ 0.05; ** P ≤ 0.01); and the P ‐values were calculated using paired ratio t ‐test. STAT 1 and STAT 2 phosphorylation in Stat1 −/− , Stat1 Y701F , Stat2 −/− , and Irf9 −/− macrophages. BMDM s were isolated from wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice and treated with 250 IU /ml of IFN β for 30 min or 6, 12, or 24 h. Whole‐cell extracts were collected and tested in Western blot for levels of phosphorylation of STAT 1 on Y701 and of STAT 2 on Y689. The same cell extracts were tested for total levels of STAT 1 and STAT 2. The blots are representative of more than three independent experiments.

    Article Snippet: ChIP was performed using STAT1 (clone E‐23, Santa Cruz) or STAT2 antibody (clone C‐20, Santa Cruz).

    Techniques: Expressing, Mouse Assay, Derivative Assay, Isolation, Western Blot, Real-time Polymerase Chain Reaction

    Presence of the STAT 1Y701F mutant reduces IFN β‐stimulated binding of STAT 2 to nuclear ISRE sequences IFN β‐stimulated binding of STAT 2 to ISRE sequences of Mx2 and Irf7 promoters. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice were treated with 250 IU /ml of IFN β for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with STAT 2‐specific antibody. The amount of precipitated DNA was measured by qPCR . Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ); asterisks denote the level of statistical significance (** P ≤ 0.01); and the P ‐values were calculated using paired ratio t ‐test. Impact of STAT 2 deficiency on IFN β‐stimulated STAT 1 association with the Mx2 ISRE . BMDM s of wild‐type ( WT ), Stat1 −/− (S1), and Stat2 −/− (S2) mice were treated with 250 IU /ml of IFN β for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with STAT 1‐specific antibody. The amount of precipitated DNA was measured by qPCR . Bars represent mean values of three independent experiments; error bars represent standard error of the mean ( SEM ). Simultaneous association of STAT 1 and STAT 2 with the Mx2 ISRE analyzed by Ch IP ‐reCh IP . BMDM s of wild‐type ( WT ) mice were treated with 250 IU /ml of IFN β for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with either STAT 1‐specific antibody and re‐immunoprecipitated with STAT 2‐specific antibody or vice versa. The amount of precipitated DNA was measured by qPCR . Bars represent mean values of three independent experiments; error bars represent standard deviation ( SD ). Impact of STAT 1Y701F on delayed, STAT 2‐mediated expression of IFN ‐induced genes. Stat1 −/− fibroblasts were transfected with plasmids driving expression of the indicated proteins. Twenty‐four hours after transfection, 250 IU /ml of IFN β was added to the transfected cells and ISG expression was determined by qPCR after 48 h of cytokine treatment. Gene expression was measured by qPCR and normalized to Gapdh. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ) and asterisks denote the level of statistical significance (* P ≤ 0.05); the P ‐values were calculated using paired ratio t ‐test.

    Journal: EMBO Reports

    Article Title: Response to interferons and antibacterial innate immunity in the absence of tyrosine‐phosphorylated STAT1

    doi: 10.15252/embr.201540726

    Figure Lengend Snippet: Presence of the STAT 1Y701F mutant reduces IFN β‐stimulated binding of STAT 2 to nuclear ISRE sequences IFN β‐stimulated binding of STAT 2 to ISRE sequences of Mx2 and Irf7 promoters. Bone marrow‐derived macrophages ( BMDM s) of wild‐type ( WT ), Stat1 Y701F ( YF ), Stat1 −/− (S1), Stat2 −/− (S2), and Irf9 −/− ( IRF 9) mice were treated with 250 IU /ml of IFN β for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with STAT 2‐specific antibody. The amount of precipitated DNA was measured by qPCR . Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ); asterisks denote the level of statistical significance (** P ≤ 0.01); and the P ‐values were calculated using paired ratio t ‐test. Impact of STAT 2 deficiency on IFN β‐stimulated STAT 1 association with the Mx2 ISRE . BMDM s of wild‐type ( WT ), Stat1 −/− (S1), and Stat2 −/− (S2) mice were treated with 250 IU /ml of IFN β for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with STAT 1‐specific antibody. The amount of precipitated DNA was measured by qPCR . Bars represent mean values of three independent experiments; error bars represent standard error of the mean ( SEM ). Simultaneous association of STAT 1 and STAT 2 with the Mx2 ISRE analyzed by Ch IP ‐reCh IP . BMDM s of wild‐type ( WT ) mice were treated with 250 IU /ml of IFN β for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with either STAT 1‐specific antibody and re‐immunoprecipitated with STAT 2‐specific antibody or vice versa. The amount of precipitated DNA was measured by qPCR . Bars represent mean values of three independent experiments; error bars represent standard deviation ( SD ). Impact of STAT 1Y701F on delayed, STAT 2‐mediated expression of IFN ‐induced genes. Stat1 −/− fibroblasts were transfected with plasmids driving expression of the indicated proteins. Twenty‐four hours after transfection, 250 IU /ml of IFN β was added to the transfected cells and ISG expression was determined by qPCR after 48 h of cytokine treatment. Gene expression was measured by qPCR and normalized to Gapdh. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean ( SEM ) and asterisks denote the level of statistical significance (* P ≤ 0.05); the P ‐values were calculated using paired ratio t ‐test.

    Article Snippet: ChIP was performed using STAT1 (clone E‐23, Santa Cruz) or STAT2 antibody (clone C‐20, Santa Cruz).

    Techniques: Mutagenesis, Binding Assay, Derivative Assay, Mouse Assay, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Transfection

    Expression of human STAT2 does not influence establishment of the murine IFN-α/β antiviral state but enhances SV5 growth in IFN-α/β-responsive murine cells. (A) Pretreatment of cells with IFN-α/β reduces efficiency of virus replication. Cell lines indicated were incubated with or without murine IFN-β (1,000 U/ml) for 6 h and then infected with 10 PFU of SV5/cell. Supernatants were harvested 48 h later and titrated by plaque assay with CV1 cells. (B) Expression of human STAT2 confers a replication advantage to SV5 in mouse cells. Cell lines were infected with SV5 at the indicated MOI, and supernatants were harvested 48 h later for virus titration. (C) Expression of human STAT2 greatly enhances SV5 replication in mouse cells in the presence of excess exogenous IFN-α/β. The experiment was conducted as for panel B but with 1,000 U of murine IFN-β/ml added at the time the SV5 inoculum was replaced, i.e., after 2 h of adsorption. (D) Replication advantage conferred by human STAT2 allows more-efficient SV5 growth during long-term infection. The methodology was similar to that described for panel B. Infected cells were supplemented with IFN-α/β as indicated (+IFN) from 2 h p.i. until day 4. Cells were passaged to larger plates at the time of harvest on day 4 and grown in the absence of IFN-α/β for the next 7 days, at the end of which time accumulated virus in the supernatant was titrated. Time line insets (all panels), experimental designs.

    Journal: Journal of Virology

    Article Title: STAT2 Acts as a Host Range Determinant for Species-Specific Paramyxovirus Interferon Antagonism and Simian Virus 5 Replication

    doi: 10.1128/JVI.76.13.6435-6441.2002

    Figure Lengend Snippet: Expression of human STAT2 does not influence establishment of the murine IFN-α/β antiviral state but enhances SV5 growth in IFN-α/β-responsive murine cells. (A) Pretreatment of cells with IFN-α/β reduces efficiency of virus replication. Cell lines indicated were incubated with or without murine IFN-β (1,000 U/ml) for 6 h and then infected with 10 PFU of SV5/cell. Supernatants were harvested 48 h later and titrated by plaque assay with CV1 cells. (B) Expression of human STAT2 confers a replication advantage to SV5 in mouse cells. Cell lines were infected with SV5 at the indicated MOI, and supernatants were harvested 48 h later for virus titration. (C) Expression of human STAT2 greatly enhances SV5 replication in mouse cells in the presence of excess exogenous IFN-α/β. The experiment was conducted as for panel B but with 1,000 U of murine IFN-β/ml added at the time the SV5 inoculum was replaced, i.e., after 2 h of adsorption. (D) Replication advantage conferred by human STAT2 allows more-efficient SV5 growth during long-term infection. The methodology was similar to that described for panel B. Infected cells were supplemented with IFN-α/β as indicated (+IFN) from 2 h p.i. until day 4. Cells were passaged to larger plates at the time of harvest on day 4 and grown in the absence of IFN-α/β for the next 7 days, at the end of which time accumulated virus in the supernatant was titrated. Time line insets (all panels), experimental designs.

    Article Snippet: Importantly, all degradation activities were observed only in cells expressing human STAT2, confirming that paramyxovirus-induced STAT degradation can utilize human STAT2 to proceed across species barriers.

    Techniques: Expressing, Incubation, Infection, Plaque Assay, Titration, Adsorption

    Expression of human STAT2 in mouse cells permits STAT degradation. NIH 3T3 cells (3T3) and a derivative NIH 3T3 line expressing human STAT2 (3T3/HuS2) were infected with 10 PFU of HPIV2 (H) or SV5 (S)/cell. Lysates prepared 16 h p.i. were separated by SDS-PAGE and processed for immunoblotting along with control uninfected cells (C). (A) Immunoblot with antiserum that recognizes HPIV2 and SV5 nucleocapsid proteins (NP SV5 ). (B) Immunoblot with antiserum specific for murine STAT2 (MuSTAT2). (C) Immunoblot with antiserum specific for murine STAT1 (MuSTAT1) that recognizes both full-length murine STAT1α and truncated STAT1β derived from alternative mRNA splicing. (D) Immunoblot with antiserum specific for human STAT2 (HuSTAT2).

    Journal: Journal of Virology

    Article Title: STAT2 Acts as a Host Range Determinant for Species-Specific Paramyxovirus Interferon Antagonism and Simian Virus 5 Replication

    doi: 10.1128/JVI.76.13.6435-6441.2002

    Figure Lengend Snippet: Expression of human STAT2 in mouse cells permits STAT degradation. NIH 3T3 cells (3T3) and a derivative NIH 3T3 line expressing human STAT2 (3T3/HuS2) were infected with 10 PFU of HPIV2 (H) or SV5 (S)/cell. Lysates prepared 16 h p.i. were separated by SDS-PAGE and processed for immunoblotting along with control uninfected cells (C). (A) Immunoblot with antiserum that recognizes HPIV2 and SV5 nucleocapsid proteins (NP SV5 ). (B) Immunoblot with antiserum specific for murine STAT2 (MuSTAT2). (C) Immunoblot with antiserum specific for murine STAT1 (MuSTAT1) that recognizes both full-length murine STAT1α and truncated STAT1β derived from alternative mRNA splicing. (D) Immunoblot with antiserum specific for human STAT2 (HuSTAT2).

    Article Snippet: Importantly, all degradation activities were observed only in cells expressing human STAT2, confirming that paramyxovirus-induced STAT degradation can utilize human STAT2 to proceed across species barriers.

    Techniques: Expressing, Infection, SDS Page, Derivative Assay

    Locations of all the ChIP–chip and expression results across chromosome 22q based on Ensembl gene annotations. The gray center track represents the regions of chromosome 22 represented on the microarray, from the centromere ( top left ) to the telomere ( bottom right ). Black bars within the gray track represent the locations of ChIP–chip results from the IFN-γ STAT1 ( top third), IFN-α–STAT1 ( middle ), and IFN-α–STAT2 ( bottom third) assays. Open diamonds and black triangles signify STAT-binding sites identified by two or three different ChIP–chip assays, respectively. Expression results from up-regulated (red), down-regulated (green), and unaffected (yellow) genes are located above (sense) and below .

    Journal: Genes & Development

    Article Title: Global changes in STAT target selection and transcription regulation upon interferon treatments

    doi: 10.1101/gad.1371305

    Figure Lengend Snippet: Locations of all the ChIP–chip and expression results across chromosome 22q based on Ensembl gene annotations. The gray center track represents the regions of chromosome 22 represented on the microarray, from the centromere ( top left ) to the telomere ( bottom right ). Black bars within the gray track represent the locations of ChIP–chip results from the IFN-γ STAT1 ( top third), IFN-α–STAT1 ( middle ), and IFN-α–STAT2 ( bottom third) assays. Open diamonds and black triangles signify STAT-binding sites identified by two or three different ChIP–chip assays, respectively. Expression results from up-regulated (red), down-regulated (green), and unaffected (yellow) genes are located above (sense) and below .

    Article Snippet: Western blotting was performed using antibodies for STAT1, STAT2, phospho-Y701-STAT1, and phospho-Y689-STAT2 (Upstate Biotechnology).

    Techniques: Chromatin Immunoprecipitation, Expressing, Microarray, Binding Assay

    IFN stimulations activate the STATs and induce their binding to correct DNA target sequences. ( A ) Western blotting of STAT1 and STAT2 immunoprecipitations from nuclear extracts detected the increase in nuclear localization and phosphorylation of STAT1 and STAT2. ( B ) PCR analysis of STAT1 and STAT2 ChIP DNA confirmed the IFN-induced enriched presence of the STAT-binding sites in the IRF1 and OAS1 promoters. Positive and negative controls used genomic DNA and no template, respectively.

    Journal: Genes & Development

    Article Title: Global changes in STAT target selection and transcription regulation upon interferon treatments

    doi: 10.1101/gad.1371305

    Figure Lengend Snippet: IFN stimulations activate the STATs and induce their binding to correct DNA target sequences. ( A ) Western blotting of STAT1 and STAT2 immunoprecipitations from nuclear extracts detected the increase in nuclear localization and phosphorylation of STAT1 and STAT2. ( B ) PCR analysis of STAT1 and STAT2 ChIP DNA confirmed the IFN-induced enriched presence of the STAT-binding sites in the IRF1 and OAS1 promoters. Positive and negative controls used genomic DNA and no template, respectively.

    Article Snippet: Western blotting was performed using antibodies for STAT1, STAT2, phospho-Y701-STAT1, and phospho-Y689-STAT2 (Upstate Biotechnology).

    Techniques: Binding Assay, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Schematic of IFN-induced STAT1-binding site selection. ( A ) IFN-γ and IFN-α both induce STAT1 homodimers, which bind DNA (Gene A); however, we also observed IFN-α-induced, STAT2-independent binding of STAT1 to sites not occupied by IFN-γ-induced STAT1 (Gene C). ( B ) Changes in binding site accessibility and/or the presence of a cooperating cofactor may account for the binding of STAT1 to new sites upon IFN-α treatment. Also, the binding of STAT1 to alternate dimerization partners (such as STAT2) reduces the relative quantity of STAT1 homodimers; thus, a loss of STAT1 binding to some IFN-γ-induced sites occurs.

    Journal: Genes & Development

    Article Title: Global changes in STAT target selection and transcription regulation upon interferon treatments

    doi: 10.1101/gad.1371305

    Figure Lengend Snippet: Schematic of IFN-induced STAT1-binding site selection. ( A ) IFN-γ and IFN-α both induce STAT1 homodimers, which bind DNA (Gene A); however, we also observed IFN-α-induced, STAT2-independent binding of STAT1 to sites not occupied by IFN-γ-induced STAT1 (Gene C). ( B ) Changes in binding site accessibility and/or the presence of a cooperating cofactor may account for the binding of STAT1 to new sites upon IFN-α treatment. Also, the binding of STAT1 to alternate dimerization partners (such as STAT2) reduces the relative quantity of STAT1 homodimers; thus, a loss of STAT1 binding to some IFN-γ-induced sites occurs.

    Article Snippet: Western blotting was performed using antibodies for STAT1, STAT2, phospho-Y701-STAT1, and phospho-Y689-STAT2 (Upstate Biotechnology).

    Techniques: Binding Assay, Selection

    The correlations between ISGF3 subunits and PBRM1, KDM5C, BAP1 or SEDT2 in the TCGA dataset. Correlations between IRF9, STAT1 or STAT2 with indicated genes within the TCGA dataset. N = 533.

    Journal: eLife

    Article Title: Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer

    doi: 10.7554/eLife.37925

    Figure Lengend Snippet: The correlations between ISGF3 subunits and PBRM1, KDM5C, BAP1 or SEDT2 in the TCGA dataset. Correlations between IRF9, STAT1 or STAT2 with indicated genes within the TCGA dataset. N = 533.

    Article Snippet: The antibodies used in this manuscript are: HA (Covance MMS-101P-1000 HA.11 (16B12 for IB, Santa Cruz Biotech sc-805 for IP), Flag (Sigma F3165-1mg (M2)), KDM5C (Bethyl A301-034A and A301-035A), BAP1 (Santa Cruz Biotech sc-28383), γ-Actin (Santa Cruz Biotech sc-8432), PBRM1 (Bethyl A301-591A and A301-590A), STAT1 (Cell signaling 9172S), phospho-STAT1 (Cell signaling 9171L), STAT2 (Bethyl A303-512A-T), phospho-STAT2 (Cell signaling 88410), IRF9 (Cell signaling 76684S), MX1 (R and D AF7946), PLSCR1 (Santa Cruz Biotech sc-59645), Vinculin (Santa Cruz Biotech sc-73614), GAPDH (Santa Cruz Biotech sc-59540), HIF2α (Novus NB100-122), OAS1 (Cell signaling 14498S).

    Techniques:

    Over-expression of IRF9 and STAT2 restored ISGF3 function and retarded tumor growth of 786-O cells with PBRM1 suppressed. ( A ) 786-O cells were infected with the indicated viral vectors. SDS-solubilized whole cell lysates were blotted with the indicated antibodies. ( B ) Cellular growth of indicated cell lines measured by WST-1 assay; ( C ) Pictures of mice and xenografted tumors and ( D ) Quantification of tumor weights originating from the same number of cancer cells expressing either an empty vector or STAT2 plus IRF9 in athymic nude mice. The p-value was calculated with two-tailed student t test.

    Journal: eLife

    Article Title: Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer

    doi: 10.7554/eLife.37925

    Figure Lengend Snippet: Over-expression of IRF9 and STAT2 restored ISGF3 function and retarded tumor growth of 786-O cells with PBRM1 suppressed. ( A ) 786-O cells were infected with the indicated viral vectors. SDS-solubilized whole cell lysates were blotted with the indicated antibodies. ( B ) Cellular growth of indicated cell lines measured by WST-1 assay; ( C ) Pictures of mice and xenografted tumors and ( D ) Quantification of tumor weights originating from the same number of cancer cells expressing either an empty vector or STAT2 plus IRF9 in athymic nude mice. The p-value was calculated with two-tailed student t test.

    Article Snippet: The antibodies used in this manuscript are: HA (Covance MMS-101P-1000 HA.11 (16B12 for IB, Santa Cruz Biotech sc-805 for IP), Flag (Sigma F3165-1mg (M2)), KDM5C (Bethyl A301-034A and A301-035A), BAP1 (Santa Cruz Biotech sc-28383), γ-Actin (Santa Cruz Biotech sc-8432), PBRM1 (Bethyl A301-591A and A301-590A), STAT1 (Cell signaling 9172S), phospho-STAT1 (Cell signaling 9171L), STAT2 (Bethyl A303-512A-T), phospho-STAT2 (Cell signaling 88410), IRF9 (Cell signaling 76684S), MX1 (R and D AF7946), PLSCR1 (Santa Cruz Biotech sc-59645), Vinculin (Santa Cruz Biotech sc-73614), GAPDH (Santa Cruz Biotech sc-59540), HIF2α (Novus NB100-122), OAS1 (Cell signaling 14498S).

    Techniques: Over Expression, Infection, WST-1 Assay, Mouse Assay, Expressing, Plasmid Preparation, Two Tailed Test

    IRF9 and STAT2 expression significantly correlated with the expression of PBRM1, SETD2, or BAP1 in ccRCC tumors. ( A ) 20x IHC images of representative ccRCC foci stained with indicated antibodies. ( B ) Spearman correlation coefficient, rho, and p-value of nuclear (nuc) IRF9 or cytoplasmic (cyto) IHC signals with those of PBRM1, SETD2 or BAP1. The analysis was performed with either all the foci or the foci within each tumor stage or tumor grade. ( C ) Kaplan-Meier curves of patient overall survival based on the positive or negative IHC signal of nuclear IRF9 and ( D ) cytoplasmic STAT2. Statistical significance calculated using the log-rank test. ( E ) A schematic diagram showing how HIF and secondary tumor suppressors converge on ISGF3 to regulate tumor growth in ccRCC. HIF activates ISGF3 that suppresses tumor growth. PBRM1, KDM5C, SETD2 and BAP1 all support the function of ISGF3. Mutations in any of them relieve the tumor suppressive function of HIF2α.

    Journal: eLife

    Article Title: Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer

    doi: 10.7554/eLife.37925

    Figure Lengend Snippet: IRF9 and STAT2 expression significantly correlated with the expression of PBRM1, SETD2, or BAP1 in ccRCC tumors. ( A ) 20x IHC images of representative ccRCC foci stained with indicated antibodies. ( B ) Spearman correlation coefficient, rho, and p-value of nuclear (nuc) IRF9 or cytoplasmic (cyto) IHC signals with those of PBRM1, SETD2 or BAP1. The analysis was performed with either all the foci or the foci within each tumor stage or tumor grade. ( C ) Kaplan-Meier curves of patient overall survival based on the positive or negative IHC signal of nuclear IRF9 and ( D ) cytoplasmic STAT2. Statistical significance calculated using the log-rank test. ( E ) A schematic diagram showing how HIF and secondary tumor suppressors converge on ISGF3 to regulate tumor growth in ccRCC. HIF activates ISGF3 that suppresses tumor growth. PBRM1, KDM5C, SETD2 and BAP1 all support the function of ISGF3. Mutations in any of them relieve the tumor suppressive function of HIF2α.

    Article Snippet: The antibodies used in this manuscript are: HA (Covance MMS-101P-1000 HA.11 (16B12 for IB, Santa Cruz Biotech sc-805 for IP), Flag (Sigma F3165-1mg (M2)), KDM5C (Bethyl A301-034A and A301-035A), BAP1 (Santa Cruz Biotech sc-28383), γ-Actin (Santa Cruz Biotech sc-8432), PBRM1 (Bethyl A301-591A and A301-590A), STAT1 (Cell signaling 9172S), phospho-STAT1 (Cell signaling 9171L), STAT2 (Bethyl A303-512A-T), phospho-STAT2 (Cell signaling 88410), IRF9 (Cell signaling 76684S), MX1 (R and D AF7946), PLSCR1 (Santa Cruz Biotech sc-59645), Vinculin (Santa Cruz Biotech sc-73614), GAPDH (Santa Cruz Biotech sc-59540), HIF2α (Novus NB100-122), OAS1 (Cell signaling 14498S).

    Techniques: Expressing, Immunohistochemistry, Staining

    STAT2 was a target of miR-506-3p, and DLX6-AS1 regulated STAT2 expression by targeting miR-506-3p. ( A ) The potential target relationship between STAT2 and miR-506-3p was predicted by the online tool Starbase3.0. ( B and C ) The potential target relationship between STAT2 and miR-506-3p was verified by dual-luciferase reporter assay. ( D and E ) The expression of STAT2 in NB tissues was detected by qRT-PCR and Western blot. ( F and G ) The expression of STAT2 in NB cells was detected by qRT-PCR and Western blot. ( H ) The correlation between STAT2 expression and miR-506-3p expression in NB tissues was analyzed according to Spearman correlation analysis. ( I and J ) The expression of STAT2 in SK-N-SH and LAN-6 cells transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA- STAT2 or miR-506-3p+pcDNA-NC was detected by qRT-PCR and Western blot at 48 h post-transfection. ( K ) The correlation between STAT2 expression and DLX6-AS1 expression in NB tissues was analyzed according to Spearman correlation analysis. ( L and M ) The expression of STAT2 in SK-N-SH and LAN-6 cells transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA-DLX6-AS1 or miR-506-3p+pcDNA-NC was examined by qRT-PCR and Western blot at 48 h post-transfection. * P

    Journal: Cancer Management and Research

    Article Title: LncRNA DLX6-AS1 Promotes the Progression of Neuroblastoma by Activating STAT2 via Targeting miR-506-3p

    doi: 10.2147/CMAR.S252521

    Figure Lengend Snippet: STAT2 was a target of miR-506-3p, and DLX6-AS1 regulated STAT2 expression by targeting miR-506-3p. ( A ) The potential target relationship between STAT2 and miR-506-3p was predicted by the online tool Starbase3.0. ( B and C ) The potential target relationship between STAT2 and miR-506-3p was verified by dual-luciferase reporter assay. ( D and E ) The expression of STAT2 in NB tissues was detected by qRT-PCR and Western blot. ( F and G ) The expression of STAT2 in NB cells was detected by qRT-PCR and Western blot. ( H ) The correlation between STAT2 expression and miR-506-3p expression in NB tissues was analyzed according to Spearman correlation analysis. ( I and J ) The expression of STAT2 in SK-N-SH and LAN-6 cells transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA- STAT2 or miR-506-3p+pcDNA-NC was detected by qRT-PCR and Western blot at 48 h post-transfection. ( K ) The correlation between STAT2 expression and DLX6-AS1 expression in NB tissues was analyzed according to Spearman correlation analysis. ( L and M ) The expression of STAT2 in SK-N-SH and LAN-6 cells transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA-DLX6-AS1 or miR-506-3p+pcDNA-NC was examined by qRT-PCR and Western blot at 48 h post-transfection. * P

    Article Snippet: Next, the membranes were probed with the primary antibodies against CDK1 (ab18; Abcam, Cambridge, MA, USA), Cyclin D1 (ab134175; Abcam), STAT2 (ab124283; Abcam), GAPDH (ab9485; Abcam) and secondary antibodies (ab205718, ab205719; Abcam).

    Techniques: Expressing, Luciferase, Reporter Assay, Quantitative RT-PCR, Western Blot, Transfection

    DLX6-AS1 deficiency inhibited NB development in vivo. Experimental mice were inoculated with SK-N-SH cells transfected with sh-DLX6-AS1 or sh-NC. ( A ) Tumor volume was measured every five days. ( B ) At 30 d post-inoculation, all tumor tissues were excised to detect tumor weight. ( C–E ) The expression of DLX6-AS1, miR-506-3p and STAT2 in excised tumor tissues was examined using qRT-PCR or Western blot. * P

    Journal: Cancer Management and Research

    Article Title: LncRNA DLX6-AS1 Promotes the Progression of Neuroblastoma by Activating STAT2 via Targeting miR-506-3p

    doi: 10.2147/CMAR.S252521

    Figure Lengend Snippet: DLX6-AS1 deficiency inhibited NB development in vivo. Experimental mice were inoculated with SK-N-SH cells transfected with sh-DLX6-AS1 or sh-NC. ( A ) Tumor volume was measured every five days. ( B ) At 30 d post-inoculation, all tumor tissues were excised to detect tumor weight. ( C–E ) The expression of DLX6-AS1, miR-506-3p and STAT2 in excised tumor tissues was examined using qRT-PCR or Western blot. * P

    Article Snippet: Next, the membranes were probed with the primary antibodies against CDK1 (ab18; Abcam, Cambridge, MA, USA), Cyclin D1 (ab134175; Abcam), STAT2 (ab124283; Abcam), GAPDH (ab9485; Abcam) and secondary antibodies (ab205718, ab205719; Abcam).

    Techniques: In Vivo, Mouse Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot

    MiR-506-3p bound to STAT2 to modulate the development of NB. SK-N-SH and LAN-6 cells were transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA- STAT2 or miR-506-3p+pcDNA-NC. ( A and B ) Cell proliferation was monitored at different time points using MTT assay. ( C and D ) Colony formation assay was performed at 48 h post-transfection. ( E and F ) Cell cycle was assessed by flow cytometry assay at 48 h post-transfection. ( G and H ) The levels of CDK1 and Cyclin D1 at 48 h post-transfection were quantified by Western blot. ( I–K ) Glucose production, lactate production and ATP level were checked at 48 h post-transfection to assess glycolysis using the corresponding kit. * P

    Journal: Cancer Management and Research

    Article Title: LncRNA DLX6-AS1 Promotes the Progression of Neuroblastoma by Activating STAT2 via Targeting miR-506-3p

    doi: 10.2147/CMAR.S252521

    Figure Lengend Snippet: MiR-506-3p bound to STAT2 to modulate the development of NB. SK-N-SH and LAN-6 cells were transfected with miR-506-3p, miR-NC, miR-506-3p+pcDNA- STAT2 or miR-506-3p+pcDNA-NC. ( A and B ) Cell proliferation was monitored at different time points using MTT assay. ( C and D ) Colony formation assay was performed at 48 h post-transfection. ( E and F ) Cell cycle was assessed by flow cytometry assay at 48 h post-transfection. ( G and H ) The levels of CDK1 and Cyclin D1 at 48 h post-transfection were quantified by Western blot. ( I–K ) Glucose production, lactate production and ATP level were checked at 48 h post-transfection to assess glycolysis using the corresponding kit. * P

    Article Snippet: Next, the membranes were probed with the primary antibodies against CDK1 (ab18; Abcam, Cambridge, MA, USA), Cyclin D1 (ab134175; Abcam), STAT2 (ab124283; Abcam), GAPDH (ab9485; Abcam) and secondary antibodies (ab205718, ab205719; Abcam).

    Techniques: Transfection, MTT Assay, Colony Assay, Flow Cytometry, Western Blot

    Phosphorylation of S734-STAT2 does not affect the antiproliferative effects of IFN-α. (A) Growth rate of U6A cells stably expressing WT-, S734A- or S734D-STAT2 was measured at 48 h and 72 h by an MTS assay. The inset shows a western blot analysis of STAT2 expression in U6A cells expressing empty vector, WT-, S734A or S734D-STAT2. (B) The same panel of cells was treated with 100 U/ml or 1000 U/ml of IFN-α for 72 h and cell viability determined by MTS assay. Results are presented as the percentage of viable cells in the presence of IFN-α compared against untreated cells. Data shown are from three or four independent experiments and presented as mean±s.e.m.

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: Phosphorylation of S734-STAT2 does not affect the antiproliferative effects of IFN-α. (A) Growth rate of U6A cells stably expressing WT-, S734A- or S734D-STAT2 was measured at 48 h and 72 h by an MTS assay. The inset shows a western blot analysis of STAT2 expression in U6A cells expressing empty vector, WT-, S734A or S734D-STAT2. (B) The same panel of cells was treated with 100 U/ml or 1000 U/ml of IFN-α for 72 h and cell viability determined by MTS assay. Results are presented as the percentage of viable cells in the presence of IFN-α compared against untreated cells. Data shown are from three or four independent experiments and presented as mean±s.e.m.

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Stable Transfection, Expressing, MTS Assay, Western Blot, Plasmid Preparation

    Subcellular localization of phosphorylated S734-STAT2. Nuclear and cytoplasmic fractions of human (A) 2fTGH and (B) hTERT cells treated with IFN-α (1000 U/ml) for the given times were evaluated for pS734-STAT2, pY-STAT2, pY-STAT1, STAT1 and STAT2 by western blot analysis as indicated. Presence of GAPDH in the cytoplasmic extract and presence of Lamin B1 in the nuclear extract confirmed purity of fractions. The arrow indicates the upper band corresponding to pS734-STAT2. Actin was used as an internal loading control.

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: Subcellular localization of phosphorylated S734-STAT2. Nuclear and cytoplasmic fractions of human (A) 2fTGH and (B) hTERT cells treated with IFN-α (1000 U/ml) for the given times were evaluated for pS734-STAT2, pY-STAT2, pY-STAT1, STAT1 and STAT2 by western blot analysis as indicated. Presence of GAPDH in the cytoplasmic extract and presence of Lamin B1 in the nuclear extract confirmed purity of fractions. The arrow indicates the upper band corresponding to pS734-STAT2. Actin was used as an internal loading control.

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Western Blot

    Mass spectrometry identifies S734 as a type I IFN-inducible phosphorylatable residue in human STAT2 that is conserved in primates. (A) Tandem mass spectrometry (MS/MS) spectrum of STAT2 after treatment with IFN-α. +P (lower panel) indicates S734 as being phosphorylated and the underlined peptide mass (lower panel) indicates the presence of the peptide in the spectrum (upper panel). P, phosphoryl group (79.97 Da). Not all peaks are annotated in the mass spectrum. Matches were made with a mass tolerance of ±2 Da. (B) S734 is mapped to the transactivation domain (TAD) of STAT2. (C) Cross-species conservation of this phosphorylation site is seen in primate STAT2.

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: Mass spectrometry identifies S734 as a type I IFN-inducible phosphorylatable residue in human STAT2 that is conserved in primates. (A) Tandem mass spectrometry (MS/MS) spectrum of STAT2 after treatment with IFN-α. +P (lower panel) indicates S734 as being phosphorylated and the underlined peptide mass (lower panel) indicates the presence of the peptide in the spectrum (upper panel). P, phosphoryl group (79.97 Da). Not all peaks are annotated in the mass spectrum. Matches were made with a mass tolerance of ±2 Da. (B) S734 is mapped to the transactivation domain (TAD) of STAT2. (C) Cross-species conservation of this phosphorylation site is seen in primate STAT2.

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Mass Spectrometry

    S734A-STAT2 enhances IFN-α-induced protection against VSV infection. (A) The U6A cell panel was left untreated or pre-treated with IFN-α (1000 U/ml) for 16 h and then infected with GFP-tagged vesicular stomatitis virus (VSV) for 36 h. Viral titers after 36 h post-viral infection (h.p.i.) were determined by plaque assay. Viral titers are shown on a log scale. (B) Western blot analysis of STAT2 expression in Stat2 −/− MEFs reconstituted with either human WT- or S734A-STAT2. (C) Stat2 −/− MEFs expressing human WT- or S734A-STAT2 were pre-treated or not with murine IFN-β (1000 U/ml) for 16 h and VSV infection was quantified by flow cytometry analysis of GFP-positive cells. Data are shown as the mean±s.e.m. from three or four independent experiments. ** P

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: S734A-STAT2 enhances IFN-α-induced protection against VSV infection. (A) The U6A cell panel was left untreated or pre-treated with IFN-α (1000 U/ml) for 16 h and then infected with GFP-tagged vesicular stomatitis virus (VSV) for 36 h. Viral titers after 36 h post-viral infection (h.p.i.) were determined by plaque assay. Viral titers are shown on a log scale. (B) Western blot analysis of STAT2 expression in Stat2 −/− MEFs reconstituted with either human WT- or S734A-STAT2. (C) Stat2 −/− MEFs expressing human WT- or S734A-STAT2 were pre-treated or not with murine IFN-β (1000 U/ml) for 16 h and VSV infection was quantified by flow cytometry analysis of GFP-positive cells. Data are shown as the mean±s.e.m. from three or four independent experiments. ** P

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Infection, Plaque Assay, Western Blot, Expressing, Flow Cytometry, Cytometry

    S734-STAT2 regulates the expression of a subset of ISGs. (A) Microarray analysis identified a small subset of ISGs for which mRNA levels were found to be enhanced in S734A-STAT2 U6A cells when compared against WT-STAT2 U6A cells after 4 h of IFN-α (1000 U/ml) treatment. Genes marked in A with an asterisk were subsequently validated by qPCR using (B) U6A or (C) Stat2 −/− MEFs reconstituted with either human WT- or S734-STAT2 treated with or without IFN-α or IFN-β (1000 U/ml) for 6, 18 or 24 h. The mRNA levels were normalized to actin expression and calculated by using ΔΔCt method. Data are presented as the mean±s.e.m. fold change from untreated cells from three or four independent experiments. * P

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: S734-STAT2 regulates the expression of a subset of ISGs. (A) Microarray analysis identified a small subset of ISGs for which mRNA levels were found to be enhanced in S734A-STAT2 U6A cells when compared against WT-STAT2 U6A cells after 4 h of IFN-α (1000 U/ml) treatment. Genes marked in A with an asterisk were subsequently validated by qPCR using (B) U6A or (C) Stat2 −/− MEFs reconstituted with either human WT- or S734-STAT2 treated with or without IFN-α or IFN-β (1000 U/ml) for 6, 18 or 24 h. The mRNA levels were normalized to actin expression and calculated by using ΔΔCt method. Data are presented as the mean±s.e.m. fold change from untreated cells from three or four independent experiments. * P

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction

    Proposed model of S734-STAT2 phosphorylation in regulating the antiviral activity of type I IFNs. Upon binding of IFN-α and IFN-β to its receptor, JAK1 and TYK2 become activated and tyrosine phosphorylate STAT1 (pY701) and STAT2 (pY690). Activated STAT1 and STAT2, together with IRF9, as the ISGF3 complex, trigger the induction of antiviral ISGs. Subsequently, tyrosine-phosphorylated STAT2 (pY690) is phosphorylated at S734 (pS734) in a JAK-dependent manner, and this in turn, restricts the induction of ISGs with antiviral activity.

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: Proposed model of S734-STAT2 phosphorylation in regulating the antiviral activity of type I IFNs. Upon binding of IFN-α and IFN-β to its receptor, JAK1 and TYK2 become activated and tyrosine phosphorylate STAT1 (pY701) and STAT2 (pY690). Activated STAT1 and STAT2, together with IRF9, as the ISGF3 complex, trigger the induction of antiviral ISGs. Subsequently, tyrosine-phosphorylated STAT2 (pY690) is phosphorylated at S734 (pS734) in a JAK-dependent manner, and this in turn, restricts the induction of ISGs with antiviral activity.

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activity Assay, Binding Assay

    STAT2 is phosphorylated at S734 in response to IFN-α treatment. (A) U6A cells stably expressing empty vector, WT-STAT2 or S734A-STAT2 were treated with human IFN-α (1000 U/ml) for the indicated times. Phosphorylation of S734-STAT2 was detected by western blot analysis using an affinity purified polyclonal antibody raised against this phosphorylated epitope. IFN-α-induced tyrosine phosphorylation of STAT2 (pY-STAT2) and STAT1 (pY-STAT1) together with total levels of STAT2 and STAT1 were also assessed by western blot analysis. (B) Immunoreactivity of the pS734-STAT2 antibody is confirmed by a lambda protein phosphatase (LPP) assay. (C–F) Immortalized hTERT cells (C) and human colorectal cancer cells F6-8 (D), T29 (E) and (F) Stat2 −/− MEFs reconstituted with human STAT2 were stimulated with either human IFN-α (1000 U/ml) or murine IFN-β (1000 U/ml), accordingly, and examined as in A. GAPDH was used as an internal loading control.

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: STAT2 is phosphorylated at S734 in response to IFN-α treatment. (A) U6A cells stably expressing empty vector, WT-STAT2 or S734A-STAT2 were treated with human IFN-α (1000 U/ml) for the indicated times. Phosphorylation of S734-STAT2 was detected by western blot analysis using an affinity purified polyclonal antibody raised against this phosphorylated epitope. IFN-α-induced tyrosine phosphorylation of STAT2 (pY-STAT2) and STAT1 (pY-STAT1) together with total levels of STAT2 and STAT1 were also assessed by western blot analysis. (B) Immunoreactivity of the pS734-STAT2 antibody is confirmed by a lambda protein phosphatase (LPP) assay. (C–F) Immortalized hTERT cells (C) and human colorectal cancer cells F6-8 (D), T29 (E) and (F) Stat2 −/− MEFs reconstituted with human STAT2 were stimulated with either human IFN-α (1000 U/ml) or murine IFN-β (1000 U/ml), accordingly, and examined as in A. GAPDH was used as an internal loading control.

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Affinity Purification

    Phosphorylation of S734-STAT2 is dependent on STAT2 tyrosine phosphorylation and catalytically active JAK kinases. (A) U6A cells stably expressing WT-, R409A/K415A (RKAA)-, or Y690F-STAT2 were left untreated or treated for 1 and 4 h with IFN-α (1000 U/ml). Tyrosine and serine phosphorylation of STAT2, and GAPDH were assessed by western blot analysis. Data were quantified and shown as the ratio of pS734-STAT2 to STAT2 and pY690-STAT2 to STAT2. The arrow indicates a non-specific protein band (NS). (B) U6A cells expressing WT-STAT2 were pre-treated or not with pan-JAK inhibitor overnight (o/n) and (C) U4A cells and U6 cells expressing WT-STAT2 stimulated with IFN-α (1000 U/ml) for the specified times were evaluated for pS734-STAT2, pY-STAT2, pY-STAT1, STAT1, STAT2 and GAPDH as indicated. (D) STAT1 immune complexes obtained from U6A cells expressing WT-STAT2 that were left untreated or treated with IFN-α (1000 U/ml) were evaluated for interactions with pS734-STAT2 and total STAT2 by western blot analysis.

    Journal: Journal of Cell Science

    Article Title: Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response

    doi: 10.1242/jcs.185421

    Figure Lengend Snippet: Phosphorylation of S734-STAT2 is dependent on STAT2 tyrosine phosphorylation and catalytically active JAK kinases. (A) U6A cells stably expressing WT-, R409A/K415A (RKAA)-, or Y690F-STAT2 were left untreated or treated for 1 and 4 h with IFN-α (1000 U/ml). Tyrosine and serine phosphorylation of STAT2, and GAPDH were assessed by western blot analysis. Data were quantified and shown as the ratio of pS734-STAT2 to STAT2 and pY690-STAT2 to STAT2. The arrow indicates a non-specific protein band (NS). (B) U6A cells expressing WT-STAT2 were pre-treated or not with pan-JAK inhibitor overnight (o/n) and (C) U4A cells and U6 cells expressing WT-STAT2 stimulated with IFN-α (1000 U/ml) for the specified times were evaluated for pS734-STAT2, pY-STAT2, pY-STAT1, STAT1, STAT2 and GAPDH as indicated. (D) STAT1 immune complexes obtained from U6A cells expressing WT-STAT2 that were left untreated or treated with IFN-α (1000 U/ml) were evaluated for interactions with pS734-STAT2 and total STAT2 by western blot analysis.

    Article Snippet: Rabbit anti-STAT2 (C-20, cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Stable Transfection, Expressing, Western Blot

    Activation of the mitochondrial dependent death pathway requires STAT2. Cells were left untreated or treated with 3,000 U/ml IFN-α for 24 and 48 h. A) Loss of mitochondrial membrane potential was measured by incubation of cells with the fluorescent

    Journal: Molecular cancer research : MCR

    Article Title: Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2

    doi: 10.1158/1541-7786.MCR-08-0344

    Figure Lengend Snippet: Activation of the mitochondrial dependent death pathway requires STAT2. Cells were left untreated or treated with 3,000 U/ml IFN-α for 24 and 48 h. A) Loss of mitochondrial membrane potential was measured by incubation of cells with the fluorescent

    Article Snippet: Antibodies directed against phospho-STAT1 Y701, STAT2, IRF-1, Cytochrome c, JAK1, TYK2, Annexin V-FITC conjugated and mouse IgG isotype FITC conjugated were from BD Biosciences/Pharmingen (Pharmingen, Palo Alto, CA).

    Techniques: Activation Assay, Incubation

    STAT2 gene silencing impairs IFN-α induced apoptosis. A) A375.S2 and SAOS2 were stably infected with shRNA lentivirus targeting STAT2 (ShRNA-STAT2), or non silencing shRNA (ShRNA-control). Percent apoptosis was measured after 72 h of IFN-α

    Journal: Molecular cancer research : MCR

    Article Title: Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2

    doi: 10.1158/1541-7786.MCR-08-0344

    Figure Lengend Snippet: STAT2 gene silencing impairs IFN-α induced apoptosis. A) A375.S2 and SAOS2 were stably infected with shRNA lentivirus targeting STAT2 (ShRNA-STAT2), or non silencing shRNA (ShRNA-control). Percent apoptosis was measured after 72 h of IFN-α

    Article Snippet: Antibodies directed against phospho-STAT1 Y701, STAT2, IRF-1, Cytochrome c, JAK1, TYK2, Annexin V-FITC conjugated and mouse IgG isotype FITC conjugated were from BD Biosciences/Pharmingen (Pharmingen, Palo Alto, CA).

    Techniques: Stable Transfection, Infection, shRNA

    Type I IFN resistant H123 cells show loss of STAT2 protein. Whole cell extracts of parental and IFN resistant H123 cells were prepared and analyzed by Western blot analysis for expression of (A) JAK1, TYK2, STAT1, and STAT3 and B) STAT2 by using antibodies

    Journal: Molecular cancer research : MCR

    Article Title: Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2

    doi: 10.1158/1541-7786.MCR-08-0344

    Figure Lengend Snippet: Type I IFN resistant H123 cells show loss of STAT2 protein. Whole cell extracts of parental and IFN resistant H123 cells were prepared and analyzed by Western blot analysis for expression of (A) JAK1, TYK2, STAT1, and STAT3 and B) STAT2 by using antibodies

    Article Snippet: Antibodies directed against phospho-STAT1 Y701, STAT2, IRF-1, Cytochrome c, JAK1, TYK2, Annexin V-FITC conjugated and mouse IgG isotype FITC conjugated were from BD Biosciences/Pharmingen (Pharmingen, Palo Alto, CA).

    Techniques: Western Blot, Expressing

    STAT2 deficient H123 cells can respond to type I IFNs and remain susceptible to other death agonists. Parental and IFN resistant H123 cells were left untreated or treated with 3,000 U/ml IFN-α for the indicated times. Whole cell extracts were

    Journal: Molecular cancer research : MCR

    Article Title: Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2

    doi: 10.1158/1541-7786.MCR-08-0344

    Figure Lengend Snippet: STAT2 deficient H123 cells can respond to type I IFNs and remain susceptible to other death agonists. Parental and IFN resistant H123 cells were left untreated or treated with 3,000 U/ml IFN-α for the indicated times. Whole cell extracts were

    Article Snippet: Antibodies directed against phospho-STAT1 Y701, STAT2, IRF-1, Cytochrome c, JAK1, TYK2, Annexin V-FITC conjugated and mouse IgG isotype FITC conjugated were from BD Biosciences/Pharmingen (Pharmingen, Palo Alto, CA).

    Techniques:

    Reconstitution of STAT2 rescues type I IFN-induced apoptosis and ISGF3 driven gene activation. (A) Percent apoptosis was measured by dually staining cells with Annexin V and PI following IFN-α stimulation. STAT2 protein levels in reconstituted

    Journal: Molecular cancer research : MCR

    Article Title: Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2

    doi: 10.1158/1541-7786.MCR-08-0344

    Figure Lengend Snippet: Reconstitution of STAT2 rescues type I IFN-induced apoptosis and ISGF3 driven gene activation. (A) Percent apoptosis was measured by dually staining cells with Annexin V and PI following IFN-α stimulation. STAT2 protein levels in reconstituted

    Article Snippet: Antibodies directed against phospho-STAT1 Y701, STAT2, IRF-1, Cytochrome c, JAK1, TYK2, Annexin V-FITC conjugated and mouse IgG isotype FITC conjugated were from BD Biosciences/Pharmingen (Pharmingen, Palo Alto, CA).

    Techniques: Activation Assay, Staining

    Nuclear localization of activated STAT2 is required for ISGF3-mediated gene expression and induction of type I IFN-induced apoptosis. STAT2 deficient H123 cells reconstituted with various forms of STAT2 were left either untreated or treated with IFN-α

    Journal: Molecular cancer research : MCR

    Article Title: Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2

    doi: 10.1158/1541-7786.MCR-08-0344

    Figure Lengend Snippet: Nuclear localization of activated STAT2 is required for ISGF3-mediated gene expression and induction of type I IFN-induced apoptosis. STAT2 deficient H123 cells reconstituted with various forms of STAT2 were left either untreated or treated with IFN-α

    Article Snippet: Antibodies directed against phospho-STAT1 Y701, STAT2, IRF-1, Cytochrome c, JAK1, TYK2, Annexin V-FITC conjugated and mouse IgG isotype FITC conjugated were from BD Biosciences/Pharmingen (Pharmingen, Palo Alto, CA).

    Techniques: Expressing

    Apigenin enhances IFN-α/β-induced JAK/STAT activation. (a) HepG2-ISRE-Luc2 cells were seeded in 96-well plates (1×10 4 /well) and treated with the various concentrations of apigenin for 2 h, then with 200 U/mL IFN-α or IFN-β for 24 h. (b) HEK293A cells were incubated with indicated concentrations of apigenin for 2 h, then with 2000 U/mL IFN-α for another 1 h. The cell lysates were immunoblotted with phospho-STAT1 (Tyr701), STAT1, phospho-STAT2 (Tyr690), or STAT2 antibodies. The quantitative results are shown. (c) HEK293A cells were treated 200 U/mL IFN-α with or without 10 µM bortezomib or the indicated concentrations of apigenin for 24 h. Real-time quantitative reverse transcription-PCR was used to determine the mRNA expression of PKR or 2’,5’-OAS1. The result is presented as induction (n-fold) relative to basal levels in untreated cells. GAPDH was used as an internal control. (*) p

    Journal: Food & Nutrition Research

    Article Title: Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation

    doi: 10.3402/fnr.v60.31288

    Figure Lengend Snippet: Apigenin enhances IFN-α/β-induced JAK/STAT activation. (a) HepG2-ISRE-Luc2 cells were seeded in 96-well plates (1×10 4 /well) and treated with the various concentrations of apigenin for 2 h, then with 200 U/mL IFN-α or IFN-β for 24 h. (b) HEK293A cells were incubated with indicated concentrations of apigenin for 2 h, then with 2000 U/mL IFN-α for another 1 h. The cell lysates were immunoblotted with phospho-STAT1 (Tyr701), STAT1, phospho-STAT2 (Tyr690), or STAT2 antibodies. The quantitative results are shown. (c) HEK293A cells were treated 200 U/mL IFN-α with or without 10 µM bortezomib or the indicated concentrations of apigenin for 24 h. Real-time quantitative reverse transcription-PCR was used to determine the mRNA expression of PKR or 2’,5’-OAS1. The result is presented as induction (n-fold) relative to basal levels in untreated cells. GAPDH was used as an internal control. (*) p

    Article Snippet: Anti-phospho-STAT1, anti-STAT1, anti-phospho-STAT2, anti-STAT2, and anti-GAPDH antibodies were purchased from Abcam (Cambridge, MA).

    Techniques: Activation Assay, Incubation, Polymerase Chain Reaction, Expressing

    Function of the NSs binding-deficient STAT2 chimera. The reporter plasmids were cotransfected into HEK293T cells with the expression plasmids for NSs and each STAT2 chimera. Twenty-four h after transfection, cells were treated with IFN-αA/D (500 U/ml) for 18 h or left untreated and then were lysed to measure luciferase activity (upper) or detect protein expression using immunoblotting (lower). The ISRE activity was calculated by dividing RLU in IFN-αA/D-treated cells by the units of cells not treated with IFN-αA/D. The ISRE activity in the absence of NSs was set as 100%. The assays were independently performed in triplicate. The data represent averages with SDs. * * , P

    Journal: Journal of Virology

    Article Title: Species-Specific Pathogenicity of Severe Fever with Thrombocytopenia Syndrome Virus Is Determined by Anti-STAT2 Activity of NSs

    doi: 10.1128/JVI.02226-18

    Figure Lengend Snippet: Function of the NSs binding-deficient STAT2 chimera. The reporter plasmids were cotransfected into HEK293T cells with the expression plasmids for NSs and each STAT2 chimera. Twenty-four h after transfection, cells were treated with IFN-αA/D (500 U/ml) for 18 h or left untreated and then were lysed to measure luciferase activity (upper) or detect protein expression using immunoblotting (lower). The ISRE activity was calculated by dividing RLU in IFN-αA/D-treated cells by the units of cells not treated with IFN-αA/D. The ISRE activity in the absence of NSs was set as 100%. The assays were independently performed in triplicate. The data represent averages with SDs. * * , P

    Article Snippet: Stat2−/− mice were purchased from the Jackson Laboratory.

    Techniques: Binding Assay, Expressing, Transfection, Luciferase, Activity Assay

    Interaction of NSs with STAT1 and STAT2. (A) HEK293T or NIH 3T3 cells were transfected with the expression plasmid for HA-tagged NSs, and co-IP assays were performed. (B) NIH 3T3 cells were transfected with the expression plasmids for HA-tagged NSs and His-tagged hSTAT2 or hamSTAT2. The protein expression levels in cell lysates (left) and in co-IP assays (right) using an anti-HA or anti-His antibody are shown. (C) Colocalization of NSs with STAT2. HEK293T, NIH 3T3, or BHK-21 cells were transfected with the expression plasmid for HA-tagged NSs and the expression plasmid for His-tagged hSTAT2, mSTAT2, or hamSTAT2, respectively. IFA was also performed with NSs, STAT2, and the nuclei shown in green, red, and blue, respectively.

    Journal: Journal of Virology

    Article Title: Species-Specific Pathogenicity of Severe Fever with Thrombocytopenia Syndrome Virus Is Determined by Anti-STAT2 Activity of NSs

    doi: 10.1128/JVI.02226-18

    Figure Lengend Snippet: Interaction of NSs with STAT1 and STAT2. (A) HEK293T or NIH 3T3 cells were transfected with the expression plasmid for HA-tagged NSs, and co-IP assays were performed. (B) NIH 3T3 cells were transfected with the expression plasmids for HA-tagged NSs and His-tagged hSTAT2 or hamSTAT2. The protein expression levels in cell lysates (left) and in co-IP assays (right) using an anti-HA or anti-His antibody are shown. (C) Colocalization of NSs with STAT2. HEK293T, NIH 3T3, or BHK-21 cells were transfected with the expression plasmid for HA-tagged NSs and the expression plasmid for His-tagged hSTAT2, mSTAT2, or hamSTAT2, respectively. IFA was also performed with NSs, STAT2, and the nuclei shown in green, red, and blue, respectively.

    Article Snippet: Stat2−/− mice were purchased from the Jackson Laboratory.

    Techniques: Transfection, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunofluorescence

    Clinical pathologies of mice infected with SFTSV. Wild-type C57BL/6 (WT), Ifnar1 −/− , Stat1 −/− , and Stat2 −/− mice were infected with 10 FFU of SFTSV (YG1). Survival (A) and body weight (B) changes were observed daily for 14 days p.i. ( n = 10). For WBC and PLT counts, blood samples were collected from two male and three female mice at 0, 1, 3, 5, and 7 days p.i. The values are shown as means ± standard deviations (SDs).

    Journal: Journal of Virology

    Article Title: Species-Specific Pathogenicity of Severe Fever with Thrombocytopenia Syndrome Virus Is Determined by Anti-STAT2 Activity of NSs

    doi: 10.1128/JVI.02226-18

    Figure Lengend Snippet: Clinical pathologies of mice infected with SFTSV. Wild-type C57BL/6 (WT), Ifnar1 −/− , Stat1 −/− , and Stat2 −/− mice were infected with 10 FFU of SFTSV (YG1). Survival (A) and body weight (B) changes were observed daily for 14 days p.i. ( n = 10). For WBC and PLT counts, blood samples were collected from two male and three female mice at 0, 1, 3, 5, and 7 days p.i. The values are shown as means ± standard deviations (SDs).

    Article Snippet: Stat2−/− mice were purchased from the Jackson Laboratory.

    Techniques: Mouse Assay, Infection

    Determination of the region of hSTAT2 important for binding to NSs. (A) Schematic representation of the chimeric mutants of hSTAT2 and mSTAT2. (B) NIH 3T3 cells were cotransfected with the expression plasmids for HA-tagged NSs and each of the STAT2 His-tagged chimeras. Representative results from the protein expression check in cell lysates (upper) and the co-IP assays using an anti-His antibody (lower) are shown. (C) IFA was also performed with NSs, STAT2, and the nuclei shown in green, red, and blue, respectively.

    Journal: Journal of Virology

    Article Title: Species-Specific Pathogenicity of Severe Fever with Thrombocytopenia Syndrome Virus Is Determined by Anti-STAT2 Activity of NSs

    doi: 10.1128/JVI.02226-18

    Figure Lengend Snippet: Determination of the region of hSTAT2 important for binding to NSs. (A) Schematic representation of the chimeric mutants of hSTAT2 and mSTAT2. (B) NIH 3T3 cells were cotransfected with the expression plasmids for HA-tagged NSs and each of the STAT2 His-tagged chimeras. Representative results from the protein expression check in cell lysates (upper) and the co-IP assays using an anti-His antibody (lower) are shown. (C) IFA was also performed with NSs, STAT2, and the nuclei shown in green, red, and blue, respectively.

    Article Snippet: Stat2−/− mice were purchased from the Jackson Laboratory.

    Techniques: Binding Assay, Expressing, Co-Immunoprecipitation Assay, Immunofluorescence

    Overexpression of STAT2 and IFNAR2 is sufficient to recapitulate differentiation-dependent changes in neuronal type I IFN-stimulated gene expression. (A) Type I IFN dose-response curves were analyzed in undifferentiated BE(2)-C (closed circles), differentiated BE(2)-C/m (closed squares), and undifferentiated cells transfected with either empty vector (open circles, solid line) or IRF-9-, IFNAR2-, and STAT2-overexpression vectors (open circles, dashed line). Normalized SEAP responses 24 h after stimulation with IFNα-A/D were fit using a variable slope nonlinear regression to calculate EC 50 and Hill slope values. (B) BE(2)-C cells were transfected with the indicated expression plasmid combinations, and EC 50 and Hill slope values were calculated from independently fit type I IFN dose-response curves as noted above in (A). * p

    Journal: PLoS ONE

    Article Title: Activation of the Type I Interferon Pathway Is Enhanced in Response to Human Neuronal Differentiation

    doi: 10.1371/journal.pone.0058813

    Figure Lengend Snippet: Overexpression of STAT2 and IFNAR2 is sufficient to recapitulate differentiation-dependent changes in neuronal type I IFN-stimulated gene expression. (A) Type I IFN dose-response curves were analyzed in undifferentiated BE(2)-C (closed circles), differentiated BE(2)-C/m (closed squares), and undifferentiated cells transfected with either empty vector (open circles, solid line) or IRF-9-, IFNAR2-, and STAT2-overexpression vectors (open circles, dashed line). Normalized SEAP responses 24 h after stimulation with IFNα-A/D were fit using a variable slope nonlinear regression to calculate EC 50 and Hill slope values. (B) BE(2)-C cells were transfected with the indicated expression plasmid combinations, and EC 50 and Hill slope values were calculated from independently fit type I IFN dose-response curves as noted above in (A). * p

    Article Snippet: Recombinant human IFNα-A/D, a hybrid universal type I IFN , was purchased from PBL Biomedical Laboratories (Piscataway, NJ) and stored as single use aliquots at −80o C. Antibodies against the indicated targets were purchased as follows: NF200 (Sigma); neuronal nuclear antigen (NeuN) and poly-sialylated neural cell adhesion molecule (PSA-NCAM; Millipore, Billerica, MA); type I IFN receptor subunit 2 (IFNAR2; PBL Biomedical Laboratories); IFN regulatory factor (IRF)-7 (Cell Signaling Technology, Danvers, MA); IRF-9 (BD Transduction Laboratories, San Jose, CA); nestin (R & D Systems); major histocompatibility complex (MHC) class I (BioLegend, San Diego, CA); signal transducer and activator of transcription (STAT) 1, STAT2, phospho-STAT1, phospho-STAT2, Tyk2, Jak1, green fluorescent protein (GFP), and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation

    Differentiation-dependent changes in type I IFN pathway expression in human BE(2)-C neuronal cells. (A) Immunoblot analysis of the indicated type I IFN pathway component or GAPDH as a loading control. Lysate from IFNα-A/D-treated BE(2)-C/m cells was used as a positive control for the known IFN-inducible components IRF-9, STAT2, and STAT1. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of untreated differentiated over undifferentiated cells. * p

    Journal: PLoS ONE

    Article Title: Activation of the Type I Interferon Pathway Is Enhanced in Response to Human Neuronal Differentiation

    doi: 10.1371/journal.pone.0058813

    Figure Lengend Snippet: Differentiation-dependent changes in type I IFN pathway expression in human BE(2)-C neuronal cells. (A) Immunoblot analysis of the indicated type I IFN pathway component or GAPDH as a loading control. Lysate from IFNα-A/D-treated BE(2)-C/m cells was used as a positive control for the known IFN-inducible components IRF-9, STAT2, and STAT1. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of untreated differentiated over undifferentiated cells. * p

    Article Snippet: Recombinant human IFNα-A/D, a hybrid universal type I IFN , was purchased from PBL Biomedical Laboratories (Piscataway, NJ) and stored as single use aliquots at −80o C. Antibodies against the indicated targets were purchased as follows: NF200 (Sigma); neuronal nuclear antigen (NeuN) and poly-sialylated neural cell adhesion molecule (PSA-NCAM; Millipore, Billerica, MA); type I IFN receptor subunit 2 (IFNAR2; PBL Biomedical Laboratories); IFN regulatory factor (IRF)-7 (Cell Signaling Technology, Danvers, MA); IRF-9 (BD Transduction Laboratories, San Jose, CA); nestin (R & D Systems); major histocompatibility complex (MHC) class I (BioLegend, San Diego, CA); signal transducer and activator of transcription (STAT) 1, STAT2, phospho-STAT1, phospho-STAT2, Tyk2, Jak1, green fluorescent protein (GFP), and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Positive Control

    Differentiation-dependent changes in type I IFN pathway function in human BE(2)-C neuronal cells. (A) Immunoblot analysis of type I IFN-induced STAT phosphorylation. Cells were treated with 5, 50, or 500 U/mL IFNα-A/D and lysates were harvested and analyzed 30 min after stimulation for phosphorylated STAT1 (p-STAT1), total STAT1, phosphorylated STAT2 (p-STAT2), and total STAT2. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of differentiated over undifferentiated cells treated with 500 U/ml IFNα-A/D. * p

    Journal: PLoS ONE

    Article Title: Activation of the Type I Interferon Pathway Is Enhanced in Response to Human Neuronal Differentiation

    doi: 10.1371/journal.pone.0058813

    Figure Lengend Snippet: Differentiation-dependent changes in type I IFN pathway function in human BE(2)-C neuronal cells. (A) Immunoblot analysis of type I IFN-induced STAT phosphorylation. Cells were treated with 5, 50, or 500 U/mL IFNα-A/D and lysates were harvested and analyzed 30 min after stimulation for phosphorylated STAT1 (p-STAT1), total STAT1, phosphorylated STAT2 (p-STAT2), and total STAT2. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of differentiated over undifferentiated cells treated with 500 U/ml IFNα-A/D. * p

    Article Snippet: Recombinant human IFNα-A/D, a hybrid universal type I IFN , was purchased from PBL Biomedical Laboratories (Piscataway, NJ) and stored as single use aliquots at −80o C. Antibodies against the indicated targets were purchased as follows: NF200 (Sigma); neuronal nuclear antigen (NeuN) and poly-sialylated neural cell adhesion molecule (PSA-NCAM; Millipore, Billerica, MA); type I IFN receptor subunit 2 (IFNAR2; PBL Biomedical Laboratories); IFN regulatory factor (IRF)-7 (Cell Signaling Technology, Danvers, MA); IRF-9 (BD Transduction Laboratories, San Jose, CA); nestin (R & D Systems); major histocompatibility complex (MHC) class I (BioLegend, San Diego, CA); signal transducer and activator of transcription (STAT) 1, STAT2, phospho-STAT1, phospho-STAT2, Tyk2, Jak1, green fluorescent protein (GFP), and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques:

    Differentiation of hESC-derived neurons enhances type I IFN pathway component expression and function. (A) Lysates from hESC-derived NPCs and mature neurons were immunoblotted for basal IRF-9, STAT1, and STAT2 expression. GAPDH was analyzed as loading control. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of mature versus immature cells. * p

    Journal: PLoS ONE

    Article Title: Activation of the Type I Interferon Pathway Is Enhanced in Response to Human Neuronal Differentiation

    doi: 10.1371/journal.pone.0058813

    Figure Lengend Snippet: Differentiation of hESC-derived neurons enhances type I IFN pathway component expression and function. (A) Lysates from hESC-derived NPCs and mature neurons were immunoblotted for basal IRF-9, STAT1, and STAT2 expression. GAPDH was analyzed as loading control. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of mature versus immature cells. * p

    Article Snippet: Recombinant human IFNα-A/D, a hybrid universal type I IFN , was purchased from PBL Biomedical Laboratories (Piscataway, NJ) and stored as single use aliquots at −80o C. Antibodies against the indicated targets were purchased as follows: NF200 (Sigma); neuronal nuclear antigen (NeuN) and poly-sialylated neural cell adhesion molecule (PSA-NCAM; Millipore, Billerica, MA); type I IFN receptor subunit 2 (IFNAR2; PBL Biomedical Laboratories); IFN regulatory factor (IRF)-7 (Cell Signaling Technology, Danvers, MA); IRF-9 (BD Transduction Laboratories, San Jose, CA); nestin (R & D Systems); major histocompatibility complex (MHC) class I (BioLegend, San Diego, CA); signal transducer and activator of transcription (STAT) 1, STAT2, phospho-STAT1, phospho-STAT2, Tyk2, Jak1, green fluorescent protein (GFP), and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Derivative Assay, Expressing

    Immunoexpression of JAK/STAT proteins in the epidermis, pemphigus vulgaris, × 400. Immunoexpression of JAK3 in the epidermis: a perilesional skin 14.38 ± 3.61; b skin lesions 18.89 ± 4.67, p > 0.05. Immunoexpression of STAT2 in the epidermis: c perilesional skin 15.79 ± 2.06; d skin lesions 17.15 ± 2.81, non-significant. Immunoexpression of STAT4 in the epidermis: e perilesional skin 24.10 ± 3.40; f skin lesions 29.08 ± 4.38, p

    Journal: Immunologic Research

    Article Title: Expression of JAK3, STAT2, STAT4, and STAT6 in pemphigus vulgaris

    doi: 10.1007/s12026-020-09122-y

    Figure Lengend Snippet: Immunoexpression of JAK/STAT proteins in the epidermis, pemphigus vulgaris, × 400. Immunoexpression of JAK3 in the epidermis: a perilesional skin 14.38 ± 3.61; b skin lesions 18.89 ± 4.67, p > 0.05. Immunoexpression of STAT2 in the epidermis: c perilesional skin 15.79 ± 2.06; d skin lesions 17.15 ± 2.81, non-significant. Immunoexpression of STAT4 in the epidermis: e perilesional skin 24.10 ± 3.40; f skin lesions 29.08 ± 4.38, p

    Article Snippet: Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in distilled water and then, sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark), and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology, Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

    Techniques:

    Morphometric analysis of JAK3 (upper left), STAT2 (upper right), STAT4 (lower left), and STAT6 (lower right) immunoexpression in keratinocytes. The results of semiquantitative analysis are expressed as the mean ± standard deviation. Control – normal skin. PV-P – pemphigus vulgaris perilesional skin, PV-L – pemphigus vulgaris skin lesions. The level of significance is defined where p

    Journal: Immunologic Research

    Article Title: Expression of JAK3, STAT2, STAT4, and STAT6 in pemphigus vulgaris

    doi: 10.1007/s12026-020-09122-y

    Figure Lengend Snippet: Morphometric analysis of JAK3 (upper left), STAT2 (upper right), STAT4 (lower left), and STAT6 (lower right) immunoexpression in keratinocytes. The results of semiquantitative analysis are expressed as the mean ± standard deviation. Control – normal skin. PV-P – pemphigus vulgaris perilesional skin, PV-L – pemphigus vulgaris skin lesions. The level of significance is defined where p

    Article Snippet: Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in distilled water and then, sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark), and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology, Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

    Techniques: Standard Deviation

    Immunoexpression of JAK/STAT proteins in the epidermis, normal skin, × 400. a Immunoexpression of JAK3 in the epidermis, normal skin, 10.73 ± 3.36. b Immunoexpression of STAT2 in the epidermis, normal skin, 11.06 ± 5.34. c Immunoexpression of STAT4 in the epidermis, normal skin, 18.59 ± 3.01. d Immunoexpression of STAT6 in the epidermis, normal skin, 11.56 ± 2.84

    Journal: Immunologic Research

    Article Title: Expression of JAK3, STAT2, STAT4, and STAT6 in pemphigus vulgaris

    doi: 10.1007/s12026-020-09122-y

    Figure Lengend Snippet: Immunoexpression of JAK/STAT proteins in the epidermis, normal skin, × 400. a Immunoexpression of JAK3 in the epidermis, normal skin, 10.73 ± 3.36. b Immunoexpression of STAT2 in the epidermis, normal skin, 11.06 ± 5.34. c Immunoexpression of STAT4 in the epidermis, normal skin, 18.59 ± 3.01. d Immunoexpression of STAT6 in the epidermis, normal skin, 11.56 ± 2.84

    Article Snippet: Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in distilled water and then, sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark), and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology, Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

    Techniques: