phospho stat1  (Cell Signaling Technology Inc)

 
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    Name:
    Stat1 Antibody
    Description:
    The Stat1 transcription factor is activated in response to a large number of ligands 1 and is essential for responsiveness to IFN α and IFN γ 2 3 Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization nuclear translocation and DNA binding 4 Stat1 protein exists as a pair of isoforms Stat1α 91 kDa and the splice variant Stat1β 84 kDa In most cells both isoforms are activated by IFN α but only Stat1α is activated by IFN γ The inappropriate activation of Stat1 occurs in many tumors 5 In addition to tyrosine phosphorylation Stat1 is also phosphorylated at Ser727 through a p38 mitogen activated protein kinase MAPK dependent pathway in response to IFN α and other cellular stresses 6 Serine phosphorylation may be required for the maximal induction of Stat1 mediated gene activation
    Catalog Number:
    9172
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human Stat1. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc phospho stat1
    Smurf1 promotes K48-linked polyubiquitination of <t>STAT1.</t> A , FLAG-STAT1 ( F-STAT1 ) and HA-Ub were cotransfected into HEK293 cells together with control vector, Myc-Smurf1(WT) ( M-Smurf1)WT) ), or Myc-Smurf1(CA) ( M-Smurf1)CA) ) expression plasmid. STAT1 ubiquitination
    The Stat1 transcription factor is activated in response to a large number of ligands 1 and is essential for responsiveness to IFN α and IFN γ 2 3 Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization nuclear translocation and DNA binding 4 Stat1 protein exists as a pair of isoforms Stat1α 91 kDa and the splice variant Stat1β 84 kDa In most cells both isoforms are activated by IFN α but only Stat1α is activated by IFN γ The inappropriate activation of Stat1 occurs in many tumors 5 In addition to tyrosine phosphorylation Stat1 is also phosphorylated at Ser727 through a p38 mitogen activated protein kinase MAPK dependent pathway in response to IFN α and other cellular stresses 6 Serine phosphorylation may be required for the maximal induction of Stat1 mediated gene activation
    https://www.bioz.com/result/phospho stat1/product/Cell Signaling Technology Inc
    Average 99 stars, based on 153 article reviews
    Price from $9.99 to $1999.99
    phospho stat1 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Smurf1 Protein Negatively Regulates Interferon-? Signaling through Promoting STAT1 Protein Ubiquitination and Degradation *"

    Article Title: Smurf1 Protein Negatively Regulates Interferon-? Signaling through Promoting STAT1 Protein Ubiquitination and Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.341198

    Smurf1 promotes K48-linked polyubiquitination of STAT1. A , FLAG-STAT1 ( F-STAT1 ) and HA-Ub were cotransfected into HEK293 cells together with control vector, Myc-Smurf1(WT) ( M-Smurf1)WT) ), or Myc-Smurf1(CA) ( M-Smurf1)CA) ) expression plasmid. STAT1 ubiquitination
    Figure Legend Snippet: Smurf1 promotes K48-linked polyubiquitination of STAT1. A , FLAG-STAT1 ( F-STAT1 ) and HA-Ub were cotransfected into HEK293 cells together with control vector, Myc-Smurf1(WT) ( M-Smurf1)WT) ), or Myc-Smurf1(CA) ( M-Smurf1)CA) ) expression plasmid. STAT1 ubiquitination

    Techniques Used: Plasmid Preparation, Expressing

    STAT1 phosphorylation is not required for Smurf1-mediated STAT1 degradation. A , FLAG-STAT1(WT) ( F-STAT1 ), FLAG-STAT1(Y701F), or FLAG-STAT1(S727A) were transfected into HEK293 together with increasing amounts of Myc-Smurf1 ( M-Smurf1 ). The protein level
    Figure Legend Snippet: STAT1 phosphorylation is not required for Smurf1-mediated STAT1 degradation. A , FLAG-STAT1(WT) ( F-STAT1 ), FLAG-STAT1(Y701F), or FLAG-STAT1(S727A) were transfected into HEK293 together with increasing amounts of Myc-Smurf1 ( M-Smurf1 ). The protein level

    Techniques Used: Transfection

    Smurf1 interacts with STAT1. A , Myc-Smurf1 ( M-Smurf1 ) and FLAG-STAT1 ( F-STAT1 ) were cotransfected into HEK293 cells, and the lysates from transfected cells were subjected to immunoprecipitation ( IP ) with anti-FLAG antibody followed by immunoblotting (
    Figure Legend Snippet: Smurf1 interacts with STAT1. A , Myc-Smurf1 ( M-Smurf1 ) and FLAG-STAT1 ( F-STAT1 ) were cotransfected into HEK293 cells, and the lysates from transfected cells were subjected to immunoprecipitation ( IP ) with anti-FLAG antibody followed by immunoblotting (

    Techniques Used: Transfection, Immunoprecipitation

    Smurf1 promotes degradation of STAT1. A , a constant amount of FLAG-STAT1 ( F-STAT1 ) expression plasmid was transfected into HEK293 cells together with increasing amounts of Smurf1 wild type or CA mutant. The protein level of STAT1 in the lysates was determined
    Figure Legend Snippet: Smurf1 promotes degradation of STAT1. A , a constant amount of FLAG-STAT1 ( F-STAT1 ) expression plasmid was transfected into HEK293 cells together with increasing amounts of Smurf1 wild type or CA mutant. The protein level of STAT1 in the lysates was determined

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Mutagenesis

    2) Product Images from "Smurf1 Protein Negatively Regulates Interferon-? Signaling through Promoting STAT1 Protein Ubiquitination and Degradation *"

    Article Title: Smurf1 Protein Negatively Regulates Interferon-? Signaling through Promoting STAT1 Protein Ubiquitination and Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.341198

    Smurf1 promotes K48-linked polyubiquitination of STAT1. A , FLAG-STAT1 ( F-STAT1 ) and HA-Ub were cotransfected into HEK293 cells together with control vector, Myc-Smurf1(WT) ( M-Smurf1)WT) ), or Myc-Smurf1(CA) ( M-Smurf1)CA) ) expression plasmid. STAT1 ubiquitination
    Figure Legend Snippet: Smurf1 promotes K48-linked polyubiquitination of STAT1. A , FLAG-STAT1 ( F-STAT1 ) and HA-Ub were cotransfected into HEK293 cells together with control vector, Myc-Smurf1(WT) ( M-Smurf1)WT) ), or Myc-Smurf1(CA) ( M-Smurf1)CA) ) expression plasmid. STAT1 ubiquitination

    Techniques Used: Plasmid Preparation, Expressing

    STAT1 phosphorylation is not required for Smurf1-mediated STAT1 degradation. A , FLAG-STAT1(WT) ( F-STAT1 ), FLAG-STAT1(Y701F), or FLAG-STAT1(S727A) were transfected into HEK293 together with increasing amounts of Myc-Smurf1 ( M-Smurf1 ). The protein level
    Figure Legend Snippet: STAT1 phosphorylation is not required for Smurf1-mediated STAT1 degradation. A , FLAG-STAT1(WT) ( F-STAT1 ), FLAG-STAT1(Y701F), or FLAG-STAT1(S727A) were transfected into HEK293 together with increasing amounts of Myc-Smurf1 ( M-Smurf1 ). The protein level

    Techniques Used: Transfection

    Smurf1 interacts with STAT1. A , Myc-Smurf1 ( M-Smurf1 ) and FLAG-STAT1 ( F-STAT1 ) were cotransfected into HEK293 cells, and the lysates from transfected cells were subjected to immunoprecipitation ( IP ) with anti-FLAG antibody followed by immunoblotting (
    Figure Legend Snippet: Smurf1 interacts with STAT1. A , Myc-Smurf1 ( M-Smurf1 ) and FLAG-STAT1 ( F-STAT1 ) were cotransfected into HEK293 cells, and the lysates from transfected cells were subjected to immunoprecipitation ( IP ) with anti-FLAG antibody followed by immunoblotting (

    Techniques Used: Transfection, Immunoprecipitation

    Smurf1 promotes degradation of STAT1. A , a constant amount of FLAG-STAT1 ( F-STAT1 ) expression plasmid was transfected into HEK293 cells together with increasing amounts of Smurf1 wild type or CA mutant. The protein level of STAT1 in the lysates was determined
    Figure Legend Snippet: Smurf1 promotes degradation of STAT1. A , a constant amount of FLAG-STAT1 ( F-STAT1 ) expression plasmid was transfected into HEK293 cells together with increasing amounts of Smurf1 wild type or CA mutant. The protein level of STAT1 in the lysates was determined

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Mutagenesis

    3) Product Images from "Epidermal Growth Factor and Interleukin-1? Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells 1"

    Article Title: Epidermal Growth Factor and Interleukin-1? Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells 1

    Journal: Biology of reproduction

    doi: 10.1095/biolreprod.104.030841

    Activation of JAK/STAT and MAP kinase signaling intermediates following challenge with IL-1β or EGF. A , B ) Cells were treated with IL-1β (10 ng/ml) or EGF (10 ng/ml) for 0–30 min and prepared for immunoblot analysis. Activation of the JAK/STAT signaling cascade was assessed by probing with antibodies recognizing total and phosphorylated forms of STAT1 (90 kDa), STAT3 (83 and 92 kDa), and STAT5 (90 kDa). Activation of the major MAP kinase signaling cascades ( B ) was assessed using antibodies recognizing total Erk-2 (42 kDa), dually phosphorylated Erk-1 (44 kDa)/Erk-2 (42 kDa), total and phosphorylated p38 (43 kDa), and total and phosphorylated JNK-1 (46 kDa)/JNK-2 (54 kDa). C ) Cells were treated with EGF and IL-1β individually or in combination for 12.5 min and prepared for immunodetection of activated signaling intermediates. These blots are representative of four independent experiments.
    Figure Legend Snippet: Activation of JAK/STAT and MAP kinase signaling intermediates following challenge with IL-1β or EGF. A , B ) Cells were treated with IL-1β (10 ng/ml) or EGF (10 ng/ml) for 0–30 min and prepared for immunoblot analysis. Activation of the JAK/STAT signaling cascade was assessed by probing with antibodies recognizing total and phosphorylated forms of STAT1 (90 kDa), STAT3 (83 and 92 kDa), and STAT5 (90 kDa). Activation of the major MAP kinase signaling cascades ( B ) was assessed using antibodies recognizing total Erk-2 (42 kDa), dually phosphorylated Erk-1 (44 kDa)/Erk-2 (42 kDa), total and phosphorylated p38 (43 kDa), and total and phosphorylated JNK-1 (46 kDa)/JNK-2 (54 kDa). C ) Cells were treated with EGF and IL-1β individually or in combination for 12.5 min and prepared for immunodetection of activated signaling intermediates. These blots are representative of four independent experiments.

    Techniques Used: Activation Assay, Immunodetection

    4) Product Images from "FC-98 Regulates TLR9-Mediated of CXCL-10 Expression in Dendritic Cells via MAPK and STAT1 Signaling Pathway"

    Article Title: FC-98 Regulates TLR9-Mediated of CXCL-10 Expression in Dendritic Cells via MAPK and STAT1 Signaling Pathway

    Journal: BioMed Research International

    doi: 10.1155/2014/926130

    FC-98 inhibited activation of MAPK and STAT1 to downregulate the CXCL-10 expression. (a) Left: BMDCs were pretreated with FC-98 for 2 h, followed by 30 min CpG treatment; the phosphorylation of of MAPK (ERK, JNK, and p38) signaling pathway was analyzed by western blot. The results shown are representative experiments from three independent assays. Right: BMDCs were cotransfected with 100 ng pAP-1 luciferase reporter plasmid and 10 ng pRL-TK-Renilla luciferase. Total amounts of plasmid DNA were equalized using empty control vector. After 24 h of culture, cells were pretreated with FC-98 for 1 h and then stimulated with 1 μ M CpG for another 6 h. Luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± SD of three independent assays. ### P
    Figure Legend Snippet: FC-98 inhibited activation of MAPK and STAT1 to downregulate the CXCL-10 expression. (a) Left: BMDCs were pretreated with FC-98 for 2 h, followed by 30 min CpG treatment; the phosphorylation of of MAPK (ERK, JNK, and p38) signaling pathway was analyzed by western blot. The results shown are representative experiments from three independent assays. Right: BMDCs were cotransfected with 100 ng pAP-1 luciferase reporter plasmid and 10 ng pRL-TK-Renilla luciferase. Total amounts of plasmid DNA were equalized using empty control vector. After 24 h of culture, cells were pretreated with FC-98 for 1 h and then stimulated with 1 μ M CpG for another 6 h. Luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± SD of three independent assays. ### P

    Techniques Used: Activation Assay, Expressing, Western Blot, Luciferase, Plasmid Preparation, Activity Assay

    5) Product Images from "Differential Production of Type I IFN Determines the Reciprocal Levels of IL-10 and Proinflammatory Cytokines Produced by C57BL/6 and BALB/c Macrophages"

    Article Title: Differential Production of Type I IFN Determines the Reciprocal Levels of IL-10 and Proinflammatory Cytokines Produced by C57BL/6 and BALB/c Macrophages

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1501923

    TLR4-dependent IFN-β production and STAT1 and IRF3 activation are higher in C57BL/6 compared with BALB/c macrophages. BMDMs were stimulated with B. pseudomallei or LPS for the indicated times. ( A ) Ifnb1 mRNA expression was determined by qRT-PCR and normalized to Hprt1 mRNA expression. ( B ) IFN-β production was quantified by ELISA. ( C ) Whole-protein extracts were generated and analyzed by Western blot for total and phosphorylated STAT1, and actin loading control. ( D ) Relative intensity of two independent experiments shown for data represented in (C). ( E ) IFN-β production was quantified by ELISA. ( F ) C57BL/6 and BALB/c macrophages were stimulated with B. pseudomallei for 2 h, and nuclear extracts were analyzed for active IRF3 by ELISA. ( G ) Whole-protein extracts were generated and analyzed by Western blot for total and phosphorylated IRF3 and heat shock protein 90 loading control. ( H ) Relative intensity of three independent experiments shown from data in (G). Graphs show means ± SEM of two to four (E) or at least three independent experiments (A and B). * p
    Figure Legend Snippet: TLR4-dependent IFN-β production and STAT1 and IRF3 activation are higher in C57BL/6 compared with BALB/c macrophages. BMDMs were stimulated with B. pseudomallei or LPS for the indicated times. ( A ) Ifnb1 mRNA expression was determined by qRT-PCR and normalized to Hprt1 mRNA expression. ( B ) IFN-β production was quantified by ELISA. ( C ) Whole-protein extracts were generated and analyzed by Western blot for total and phosphorylated STAT1, and actin loading control. ( D ) Relative intensity of two independent experiments shown for data represented in (C). ( E ) IFN-β production was quantified by ELISA. ( F ) C57BL/6 and BALB/c macrophages were stimulated with B. pseudomallei for 2 h, and nuclear extracts were analyzed for active IRF3 by ELISA. ( G ) Whole-protein extracts were generated and analyzed by Western blot for total and phosphorylated IRF3 and heat shock protein 90 loading control. ( H ) Relative intensity of three independent experiments shown from data in (G). Graphs show means ± SEM of two to four (E) or at least three independent experiments (A and B). * p

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Generated, Western Blot

    6) Product Images from "Metabolic control of BRISC-SHMT2 assembly regulates immune signaling"

    Article Title: Metabolic control of BRISC-SHMT2 assembly regulates immune signaling

    Journal: Nature

    doi: 10.1038/s41586-019-1232-1

    BRISC-SHMT2 interaction is important for interferon signaling a b) Immunoprecipitation (IP) was performed using anti-Flag antibody in 293T cells transiently transfected with Flag-HA epitope-tagged Abraxas2 wild-type (WT) or mutants. Immunoblotting was performed for the indicated proteins. c) Abraxas2 –/– MEFs and Abraxas2 –/– MEFs stably reconstituted with WT or mutants were infected with Herpes Simplex Virus (HSV) lacking the lytic phase gene ICP0. IFN signalling was assessed by immunoblotting for STAT1 phosphorylated at Y701 (pSTAT1). d) WT MEFs overexpressing Abraxas2 WT or E144R were challenged with LPS and IFNβ. IFN signaling was assessed as in c . Data in a - d are representative of three independent experiments. For gel source data, see Supplementary Figure 1 . e) Volcano plots illustrating the fold change in gene expression of IFN type I-stimulated genes relative to Abraxas2 –/– KO MEFs without LPS treatment. p-values were calculated using a Student’s t-test (two-tail distribution and equal variances between the two samples) on the triplicate 2^(-ΔCT) values for each gene in each treatment group compared to the control group. Genes below the p=0.05 threshold were upregulated in WT+LPS, but represented as below statistical significance due to the relative expression in KO samples being almost zero. Regulated genes are highlighted in Extended Data Fig. 9a .
    Figure Legend Snippet: BRISC-SHMT2 interaction is important for interferon signaling a b) Immunoprecipitation (IP) was performed using anti-Flag antibody in 293T cells transiently transfected with Flag-HA epitope-tagged Abraxas2 wild-type (WT) or mutants. Immunoblotting was performed for the indicated proteins. c) Abraxas2 –/– MEFs and Abraxas2 –/– MEFs stably reconstituted with WT or mutants were infected with Herpes Simplex Virus (HSV) lacking the lytic phase gene ICP0. IFN signalling was assessed by immunoblotting for STAT1 phosphorylated at Y701 (pSTAT1). d) WT MEFs overexpressing Abraxas2 WT or E144R were challenged with LPS and IFNβ. IFN signaling was assessed as in c . Data in a - d are representative of three independent experiments. For gel source data, see Supplementary Figure 1 . e) Volcano plots illustrating the fold change in gene expression of IFN type I-stimulated genes relative to Abraxas2 –/– KO MEFs without LPS treatment. p-values were calculated using a Student’s t-test (two-tail distribution and equal variances between the two samples) on the triplicate 2^(-ΔCT) values for each gene in each treatment group compared to the control group. Genes below the p=0.05 threshold were upregulated in WT+LPS, but represented as below statistical significance due to the relative expression in KO samples being almost zero. Regulated genes are highlighted in Extended Data Fig. 9a .

    Techniques Used: Immunoprecipitation, Transfection, Stable Transfection, Infection, Expressing

    Purification and analysis of SHMT2 mutants a) Elution profile of the indicated SHMT2ΔN forms from an S75 10/300 size exclusion chromatography column (single experiment). b) Coomassie-stained SDS-PAGE analysis of the indicated SHMT2ΔN protein preparations (data are representative of two independent experiments). c) BRISC DUB activity against a fluorogenic K63-linked diUb substrate in the presence of the indicated SHMT2ΔN mutants. Data are average +/-SEM of three independent experiments carried out in duplicate. d) BRISC DUB activity against K63-linked hexa-Ub chains in the presence of the SHMT2ΔN or SHMT1 forms. e) Ubiquitylation levels of IFNAR1 after IFNα stimulation in 293T cells overexpressing the indicated Abraxas2 and SHMT2ΔN forms (LL- > RR = L211R+L215R). IFNAR1 immunoprecipitation (IP) was performed under denaturing conditions and ubiquitin levels were detected using the vu-1 antibody. Mock IP was performed using a generic rabbit IgG antibody. f) Immunoprecipitation (IP) performed using anti-Flag antibody in MEFs transiently transfected with Flag-HA epitope-tagged SHMT2ΔN or mutants. Immunoblot was performed for Abraxas2 and SHMT2 as indicated. UTF = untransfected cells used as control. g) MEFs overexpressing the indicated SHMT2ΔN or mutants were challenged with LPS and interferon receptor-dependent signal transduction response was assessed by immunoblot for STAT1 phosphorylated at Y701 (pSTAT1). Data shown in d - e are representative of three independent experiments. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: Purification and analysis of SHMT2 mutants a) Elution profile of the indicated SHMT2ΔN forms from an S75 10/300 size exclusion chromatography column (single experiment). b) Coomassie-stained SDS-PAGE analysis of the indicated SHMT2ΔN protein preparations (data are representative of two independent experiments). c) BRISC DUB activity against a fluorogenic K63-linked diUb substrate in the presence of the indicated SHMT2ΔN mutants. Data are average +/-SEM of three independent experiments carried out in duplicate. d) BRISC DUB activity against K63-linked hexa-Ub chains in the presence of the SHMT2ΔN or SHMT1 forms. e) Ubiquitylation levels of IFNAR1 after IFNα stimulation in 293T cells overexpressing the indicated Abraxas2 and SHMT2ΔN forms (LL- > RR = L211R+L215R). IFNAR1 immunoprecipitation (IP) was performed under denaturing conditions and ubiquitin levels were detected using the vu-1 antibody. Mock IP was performed using a generic rabbit IgG antibody. f) Immunoprecipitation (IP) performed using anti-Flag antibody in MEFs transiently transfected with Flag-HA epitope-tagged SHMT2ΔN or mutants. Immunoblot was performed for Abraxas2 and SHMT2 as indicated. UTF = untransfected cells used as control. g) MEFs overexpressing the indicated SHMT2ΔN or mutants were challenged with LPS and interferon receptor-dependent signal transduction response was assessed by immunoblot for STAT1 phosphorylated at Y701 (pSTAT1). Data shown in d - e are representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Purification, Size-exclusion Chromatography, Staining, SDS Page, Activity Assay, Immunoprecipitation, Transfection, Transduction

    7) Product Images from "STAT1-HDAC4 signaling induces epithelial-mesenchymal transition and sphere formation of cancer cells overexpressing the oncogene, CUG2"

    Article Title: STAT1-HDAC4 signaling induces epithelial-mesenchymal transition and sphere formation of cancer cells overexpressing the oncogene, CUG2

    Journal: Oncology Reports

    doi: 10.3892/or.2018.6701

    STAT1 silencing inhibits CUG2-induced cell migration and invasion. (A) Cell migration was measured by a wound healing assay at 48 h post-transfection with STAT1 siRNA (500 nM) or control siRNA. (B) An invasion assay was performed using 48-well Boyden chambers coated with Matrigel at 48 h post-transfection with STAT1 siRNA or control siRNA. Scale bar indicates 100 µm (***P
    Figure Legend Snippet: STAT1 silencing inhibits CUG2-induced cell migration and invasion. (A) Cell migration was measured by a wound healing assay at 48 h post-transfection with STAT1 siRNA (500 nM) or control siRNA. (B) An invasion assay was performed using 48-well Boyden chambers coated with Matrigel at 48 h post-transfection with STAT1 siRNA or control siRNA. Scale bar indicates 100 µm (***P

    Techniques Used: Migration, Wound Healing Assay, Transfection, Invasion Assay

    Constitutive suppression of STAT1 inhibits CUG2-induced cell migration, invasion, and sphere formation. (A) After transfection with sh-STAT1 (A549-CUG2-shSTAT1) or control plasmid (A549-CUG2-shVec) and selection under puromycin (1 µg/ml), suppression of STAT1 expression was confirmed by immunoblotting using an anti-STAT1 antibody. (B) After confluence of A549-CUG2-shSTAT1 and A549-CUG2-shVec cells, the cell monolayer was scratched. Cell migration was measured by a wound healing assay. (C) An invasion assay was compared between A549-CUG2-shSTAT1 and A549-CUG2-shVec cells using 48-well Boyden chambers coated with Matrigel. Scale bar indicates 100 µm (***P
    Figure Legend Snippet: Constitutive suppression of STAT1 inhibits CUG2-induced cell migration, invasion, and sphere formation. (A) After transfection with sh-STAT1 (A549-CUG2-shSTAT1) or control plasmid (A549-CUG2-shVec) and selection under puromycin (1 µg/ml), suppression of STAT1 expression was confirmed by immunoblotting using an anti-STAT1 antibody. (B) After confluence of A549-CUG2-shSTAT1 and A549-CUG2-shVec cells, the cell monolayer was scratched. Cell migration was measured by a wound healing assay. (C) An invasion assay was compared between A549-CUG2-shSTAT1 and A549-CUG2-shVec cells using 48-well Boyden chambers coated with Matrigel. Scale bar indicates 100 µm (***P

    Techniques Used: Migration, Transfection, Plasmid Preparation, Selection, Expressing, Wound Healing Assay, Invasion Assay

    STAT1 silencing inhibits expression of stemness-related factors and sphere formation. (A) After transfection with STAT1 siRNA or control siRNA, spheroid size and number were evaluated at 2, 4 and 6 days post-seeding. A spheroid greater than 50 µm in size was the criterion for evaluating sphere formation. Scale bars indicate 50 µm (***P
    Figure Legend Snippet: STAT1 silencing inhibits expression of stemness-related factors and sphere formation. (A) After transfection with STAT1 siRNA or control siRNA, spheroid size and number were evaluated at 2, 4 and 6 days post-seeding. A spheroid greater than 50 µm in size was the criterion for evaluating sphere formation. Scale bars indicate 50 µm (***P

    Techniques Used: Expressing, Transfection

    8) Product Images from "Protein Inhibitor of Activated STAT Y (PIASy) and a Splice Variant Lacking Exon 6 Enhance Sumoylation but Are Not Essential for Embryogenesis and Adult Life"

    Article Title: Protein Inhibitor of Activated STAT Y (PIASy) and a Splice Variant Lacking Exon 6 Enhance Sumoylation but Are Not Essential for Embryogenesis and Adult Life

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.12.5577-5586.2004

    No major differences in STAT1 activation in Piasy −/− primary splenocytes. (A) Western blots for STAT1 tyrosine phosphorylation upon IFN-γ treatment in +/+ and −/− splenocytes. Top panel, blotted for phospho-STAT1; bottom panel, blotted for STAT1. Cells were untreated or pulsed with IFN-γ (20 min) and then washed and incubated for the indicated time in minutes before lysis. (B) Northern analysis of IFN-γ-induced gene induction of IRF-1 and SOCS-1 from Piasy +/+ and Piasy −/− splenocytes over 60 min with quantitative bar graphs normalized to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels below. The time of stimulation in minutes is indicated across the top. The probes used are indicated to the right of Northern blots. WT, wild type; KO, knockout.
    Figure Legend Snippet: No major differences in STAT1 activation in Piasy −/− primary splenocytes. (A) Western blots for STAT1 tyrosine phosphorylation upon IFN-γ treatment in +/+ and −/− splenocytes. Top panel, blotted for phospho-STAT1; bottom panel, blotted for STAT1. Cells were untreated or pulsed with IFN-γ (20 min) and then washed and incubated for the indicated time in minutes before lysis. (B) Northern analysis of IFN-γ-induced gene induction of IRF-1 and SOCS-1 from Piasy +/+ and Piasy −/− splenocytes over 60 min with quantitative bar graphs normalized to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels below. The time of stimulation in minutes is indicated across the top. The probes used are indicated to the right of Northern blots. WT, wild type; KO, knockout.

    Techniques Used: Activation Assay, Western Blot, Incubation, Lysis, Northern Blot, Knock-Out

    9) Product Images from "JAK2 V617F stimulates proliferation of erythropoietin- dependent erythroid progenitors and delays their differentiation by activating Stat1 and other non-erythroid signaling pathways"

    Article Title: JAK2 V617F stimulates proliferation of erythropoietin- dependent erythroid progenitors and delays their differentiation by activating Stat1 and other non-erythroid signaling pathways

    Journal: Experimental hematology

    doi: 10.1016/j.exphem.2016.07.010

    Activation of non-erythroid signaling pathways in erythroid progenitors expressing JAK2 V617F ) (b) Western blots show expression of STAT1, STAT3, STAT5 and STAT6 in control, JAK2-expressing, and JAK2 V617F-expressing erythroid progenitors at 2.5 hours after Epo simulation. Strikingly, hyper expression and activation (phosphorylation) of STAT1, STAT3 and STAT5 were only found in progenitors expressing JAK2 V617F. Moreover, Western blots show expression of several cytokine receptors, including Glycoprotein 130 (GP130), cytokine receptor common beta chain (Csf2rb), Interferon gamma receptor 1(IFNGR1) and chemokine (C-X-C motif) receptor 4 (CXCR4) in erythroid progenitors at 2.5 hours culture after Epo simulation. Importantly, phosphorylation of IFNGR1 and CXCR4 were only found in progenitors expressing JAK2 V617F. GAPDH was used as loading control.
    Figure Legend Snippet: Activation of non-erythroid signaling pathways in erythroid progenitors expressing JAK2 V617F ) (b) Western blots show expression of STAT1, STAT3, STAT5 and STAT6 in control, JAK2-expressing, and JAK2 V617F-expressing erythroid progenitors at 2.5 hours after Epo simulation. Strikingly, hyper expression and activation (phosphorylation) of STAT1, STAT3 and STAT5 were only found in progenitors expressing JAK2 V617F. Moreover, Western blots show expression of several cytokine receptors, including Glycoprotein 130 (GP130), cytokine receptor common beta chain (Csf2rb), Interferon gamma receptor 1(IFNGR1) and chemokine (C-X-C motif) receptor 4 (CXCR4) in erythroid progenitors at 2.5 hours culture after Epo simulation. Importantly, phosphorylation of IFNGR1 and CXCR4 were only found in progenitors expressing JAK2 V617F. GAPDH was used as loading control.

    Techniques Used: Activation Assay, Expressing, Western Blot

    Inhibition of Stat1 disrupted the erythroid hyper-proliferation of cells expressing JAK2 V617F (a) A Stat1 inhibitor, Fludorabine, blocked the proliferation of cells expressing JAK2 V617F, leading to a significant decrease in cell numbers during differentiation, while the Stat3 inhibitor, Stattic, only moderately disturbed the proliferation of JAK2 V617F cells, comparing to control cells treated with DMSO. (b) Compared with cells treated with DMSO and Stattic, more cells treated with Fludorabine were Annexin V positive, suggesting more cells underwent apoptosis. (c) Western blots showed the Stat1 phosphorylation decreased in cells treated with Fludorabine, while Stat3 phosphorylation was also slightly reduced in cells treated with Stattic, comparing to cells treated with DMSO. Western blot quantifications were done by Image J. The degree of Stat1 and Stat3 phosphorylation were represented by the rations of pStat1 to Stat1 and pStat3 to Stat3, respectively, which were normalized to those in cells treated with DMSO (set to 1.0). (*: p
    Figure Legend Snippet: Inhibition of Stat1 disrupted the erythroid hyper-proliferation of cells expressing JAK2 V617F (a) A Stat1 inhibitor, Fludorabine, blocked the proliferation of cells expressing JAK2 V617F, leading to a significant decrease in cell numbers during differentiation, while the Stat3 inhibitor, Stattic, only moderately disturbed the proliferation of JAK2 V617F cells, comparing to control cells treated with DMSO. (b) Compared with cells treated with DMSO and Stattic, more cells treated with Fludorabine were Annexin V positive, suggesting more cells underwent apoptosis. (c) Western blots showed the Stat1 phosphorylation decreased in cells treated with Fludorabine, while Stat3 phosphorylation was also slightly reduced in cells treated with Stattic, comparing to cells treated with DMSO. Western blot quantifications were done by Image J. The degree of Stat1 and Stat3 phosphorylation were represented by the rations of pStat1 to Stat1 and pStat3 to Stat3, respectively, which were normalized to those in cells treated with DMSO (set to 1.0). (*: p

    Techniques Used: Inhibition, Expressing, Western Blot

    10) Product Images from "Different STAT transcription complexes drive early and delayed responses to type I Interferons"

    Article Title: Different STAT transcription complexes drive early and delayed responses to type I Interferons

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1401139

    Jak1 is activated with delayed kinetics in IFN-I stimulated Stat1[−/−] BMMs
    Figure Legend Snippet: Jak1 is activated with delayed kinetics in IFN-I stimulated Stat1[−/−] BMMs

    Techniques Used:

    L. pneumophila growth in Stat1[−/−] and Stat2[−/−] BMMs
    Figure Legend Snippet: L. pneumophila growth in Stat1[−/−] and Stat2[−/−] BMMs

    Techniques Used:

    IFN-Is activate a novel ISRE binding complex in Stat1[−/−] BMMs
    Figure Legend Snippet: IFN-Is activate a novel ISRE binding complex in Stat1[−/−] BMMs

    Techniques Used: Binding Assay

    IRF-9 is required for IFN-I stimulated ISG expression in Stat1[−/−] BMMs
    Figure Legend Snippet: IRF-9 is required for IFN-I stimulated ISG expression in Stat1[−/−] BMMs

    Techniques Used: Expressing

    11) Product Images from "Constitutive metanephric mesenchyme-specific expression of interferon-gamma causes renal dysplasia by regulating Sall1 expression"

    Article Title: Constitutive metanephric mesenchyme-specific expression of interferon-gamma causes renal dysplasia by regulating Sall1 expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197356

    Expression of Ifng increases in the kidneys of the Ifng GOF mouse. A. Schematic depicting the Ifng GOF mouse line. Murine Ifng is ectopically expressed in the Pax3-expressing domain after Cre-mediated excision of a LoxP-CAT-Stop cassette. B. The amount of Ifng in kidneys increases about 13 fold in mutant vs. normal kidneys, when Ifng expression is targeted to the MM. The amount of Ifng was measured by ELISA. C. Ifng expression (red) is elevated in the MM-derived areas. The UB is marked with Hoxb7/myr-venus (green). Scale bar = 20 μm. D. Ifng mRNA is elevated in kidneys from Ifng GOF mice. mRNA is measured by semi-quantitative RT-PCR. Ifngr1 and Ifngr2 are also detected in mouse embryonic kidneys. E. Stat1 protein is activated in mutant kidneys, as determined by phosphorylation of Y701 and S727 in immunoblots. As a loading control, β-Actin was used. All these experiments used E14.5 kidneys. nor—normal, mu—mutant.
    Figure Legend Snippet: Expression of Ifng increases in the kidneys of the Ifng GOF mouse. A. Schematic depicting the Ifng GOF mouse line. Murine Ifng is ectopically expressed in the Pax3-expressing domain after Cre-mediated excision of a LoxP-CAT-Stop cassette. B. The amount of Ifng in kidneys increases about 13 fold in mutant vs. normal kidneys, when Ifng expression is targeted to the MM. The amount of Ifng was measured by ELISA. C. Ifng expression (red) is elevated in the MM-derived areas. The UB is marked with Hoxb7/myr-venus (green). Scale bar = 20 μm. D. Ifng mRNA is elevated in kidneys from Ifng GOF mice. mRNA is measured by semi-quantitative RT-PCR. Ifngr1 and Ifngr2 are also detected in mouse embryonic kidneys. E. Stat1 protein is activated in mutant kidneys, as determined by phosphorylation of Y701 and S727 in immunoblots. As a loading control, β-Actin was used. All these experiments used E14.5 kidneys. nor—normal, mu—mutant.

    Techniques Used: Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Derivative Assay, Mouse Assay, Quantitative RT-PCR, Western Blot

    12) Product Images from "Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation"

    Article Title: Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26088-y

    NTG-A-009 reduces Th1 and Th17 cells differentiation through the inhibition of JAK/STAT pathway. ( a ) Naïve CD4 + T cells isolated from spleen and lympnodes were cultured with or without NTG-A-009 under Th1 condition and re stimulated with anti-CD3 (1 μg/ml) and anti-CD28(1 μg/ml) followed by the analysis of the expression of indicated proteins in JAK/STAT pathway by western blot. Phosphorylated and total form of STAT1, STAT4, and JAK1/2 were analyzed by immunoblotting under Th1 condition. ( b ) Phosphorylated and total form of STAT3 and JAK1/2 were detected by immunoblotting under Th17 condition. Full length blots and gels were shown in supplementary information- 7, 8, 9 and 10. Data are the representative of three independent experiments.
    Figure Legend Snippet: NTG-A-009 reduces Th1 and Th17 cells differentiation through the inhibition of JAK/STAT pathway. ( a ) Naïve CD4 + T cells isolated from spleen and lympnodes were cultured with or without NTG-A-009 under Th1 condition and re stimulated with anti-CD3 (1 μg/ml) and anti-CD28(1 μg/ml) followed by the analysis of the expression of indicated proteins in JAK/STAT pathway by western blot. Phosphorylated and total form of STAT1, STAT4, and JAK1/2 were analyzed by immunoblotting under Th1 condition. ( b ) Phosphorylated and total form of STAT3 and JAK1/2 were detected by immunoblotting under Th17 condition. Full length blots and gels were shown in supplementary information- 7, 8, 9 and 10. Data are the representative of three independent experiments.

    Techniques Used: Inhibition, Isolation, Cell Culture, Expressing, Western Blot

    13) Product Images from "Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ"

    Article Title: Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.01.002

    IDO expression in IFN-γ-primed MSCs via a JAK-STAT1 signaling pathway. MSCs derived from four different tissues (BM-, AT-, CB-, and WJ-MSC) were used. (a) MSCs were incubated with 200 IU/mL IFN-γ for the indicated amounts of time. The expression levels of phospho-JAK1/2, phospho-STAT1, STAT1, and IRF-1 in these MSCs were detected by immunoblotting. (b) To inhibit the activity of JAK, an intracellular domain of the IFN-γ receptor, MSCs were incubated with 1 μM AG490 (a JAK inhibitor) for 24 h before IFN-γ priming. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in AG490-treated MSCs were detected by immunoblotting. AG490 treatment induced the down-regulation of STAT1 activity and IDO expression. (c) To down-regulate STAT1 activity, MSCs were transfected with a scrambled siRNA or with an siRNA targeting STAT1 . The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these transfected MSCs were detected by immunoblotting. Down-regulation of STAT1 activity effectively induced a decrease in IDO expression in IFN-γ-primed MSCs. (d) MSCs were treated with 200 IU/mL IFN-γ or 100 μg/mL poly I:C for 24 h. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these MSCs were detected by immunoblotting. β-Actin was used as a loading control for all western blots.
    Figure Legend Snippet: IDO expression in IFN-γ-primed MSCs via a JAK-STAT1 signaling pathway. MSCs derived from four different tissues (BM-, AT-, CB-, and WJ-MSC) were used. (a) MSCs were incubated with 200 IU/mL IFN-γ for the indicated amounts of time. The expression levels of phospho-JAK1/2, phospho-STAT1, STAT1, and IRF-1 in these MSCs were detected by immunoblotting. (b) To inhibit the activity of JAK, an intracellular domain of the IFN-γ receptor, MSCs were incubated with 1 μM AG490 (a JAK inhibitor) for 24 h before IFN-γ priming. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in AG490-treated MSCs were detected by immunoblotting. AG490 treatment induced the down-regulation of STAT1 activity and IDO expression. (c) To down-regulate STAT1 activity, MSCs were transfected with a scrambled siRNA or with an siRNA targeting STAT1 . The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these transfected MSCs were detected by immunoblotting. Down-regulation of STAT1 activity effectively induced a decrease in IDO expression in IFN-γ-primed MSCs. (d) MSCs were treated with 200 IU/mL IFN-γ or 100 μg/mL poly I:C for 24 h. The expression levels of phospho-STAT1, STAT1, IDO, and IRF-1 in these MSCs were detected by immunoblotting. β-Actin was used as a loading control for all western blots.

    Techniques Used: Expressing, Derivative Assay, Incubation, Activity Assay, Transfection, Western Blot

    Enhanced immunosuppressive properties of IFN-γ-primed MSCs. (a–b) 200 IU/mL IFN-γ was added to MSCs, and the cells were incubated for 24 h. PHA-stimulated hPBMCs were incubated in the absence or presence of PBS-treated, or IFN-γ-primed MSCs. (a) Pretreatment with an anti-IFN-γ antibody (#1, once; #2, twice) before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. (b) Down-regulation of STAT1 activity using an siRNA before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. hPBMC proliferation was evaluated on day 3 and is expressed as the percentage of BrdU + cells. Data are expressed as the percentage of hPBMC proliferation in the absence of MSCs and represent the mean ± SD of three separate experiments. **P
    Figure Legend Snippet: Enhanced immunosuppressive properties of IFN-γ-primed MSCs. (a–b) 200 IU/mL IFN-γ was added to MSCs, and the cells were incubated for 24 h. PHA-stimulated hPBMCs were incubated in the absence or presence of PBS-treated, or IFN-γ-primed MSCs. (a) Pretreatment with an anti-IFN-γ antibody (#1, once; #2, twice) before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. (b) Down-regulation of STAT1 activity using an siRNA before IFN-γ priming significantly decreased the suppressive effect of MSCs on PHA-induced T-cell proliferation. hPBMC proliferation was evaluated on day 3 and is expressed as the percentage of BrdU + cells. Data are expressed as the percentage of hPBMC proliferation in the absence of MSCs and represent the mean ± SD of three separate experiments. **P

    Techniques Used: Incubation, Activity Assay

    14) Product Images from "Interferon-? Regulates Glutaminase 1 Promoter through STAT1 Phosphorylation: Relevance to HIV-1 Associated Neurocognitive Disorders"

    Article Title: Interferon-? Regulates Glutaminase 1 Promoter through STAT1 Phosphorylation: Relevance to HIV-1 Associated Neurocognitive Disorders

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032995

    STAT1 binds directly with the GLS1 promoter in IFN-α treated MDM. (A). The predicted STAT1 binding sites in the human GLS1 promoter, TSS is designated as +1. (B). MDM were treated with 100 U/ml IFN-α for 1 hour, then ChIP assay was performed using digested chromatin, p-STAT1 (Tyr 701) and STAT1 antibodies, or IgG antibody as a negative control. Purified DNA was analyzed by quantitative real-time PCR using specific primers. The amount of immunoprecipitated DNA is represented as signal relative to the total amount of input chromatin. The data are representative of three independent experiments using three different donors. #, p
    Figure Legend Snippet: STAT1 binds directly with the GLS1 promoter in IFN-α treated MDM. (A). The predicted STAT1 binding sites in the human GLS1 promoter, TSS is designated as +1. (B). MDM were treated with 100 U/ml IFN-α for 1 hour, then ChIP assay was performed using digested chromatin, p-STAT1 (Tyr 701) and STAT1 antibodies, or IgG antibody as a negative control. Purified DNA was analyzed by quantitative real-time PCR using specific primers. The amount of immunoprecipitated DNA is represented as signal relative to the total amount of input chromatin. The data are representative of three independent experiments using three different donors. #, p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Negative Control, Purification, Real-time Polymerase Chain Reaction, Immunoprecipitation

    STAT1 binds directly with the GLS1 promoter in HIV-1 infected MDM. (A-E). MDM were infected with or without HIV-1 ADA for 5 days. (A). ChIP was performed using p-STAT1 (Tyr 701) antibody; IgG was used as a negative control. (B). Supernatants were tested for RTase activity. (C). p-STAT1 and STAT1 were detected by Western blot with β-actin used as a loading control. (D). Glutamate was detected in supernatants by HPLC. (E). Intracellular glutamate was detected using the Amplex ® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data (A-E) are representative of three independent experiments using three different donors. (F) Human MDM were infected with HIV-1 for 5 days, then total RNA was collected. IFN-α and IFN-β mRNA levels were determined by real-time RT-PCR. Results shown are representative of three independent experiments using three different donors and are means of triplicate samples. (G-I) Human MDM were infected with HIV-1 for 5 days in the presence of IgG or IFN-α/IFN-β neutralizing antibodies. Total RNA and cell lysates were collected and GAC mRNA (G) and protein (H and I) levels were determined by real time RT-PCR and Western blot, respectively. For quantification, GAC expression were normalized as a ratio to β-actin and shown as fold change relative to IgG control . Results (F, I) are shown as the average ± SEM of three independent experiments with three different donors, #, p
    Figure Legend Snippet: STAT1 binds directly with the GLS1 promoter in HIV-1 infected MDM. (A-E). MDM were infected with or without HIV-1 ADA for 5 days. (A). ChIP was performed using p-STAT1 (Tyr 701) antibody; IgG was used as a negative control. (B). Supernatants were tested for RTase activity. (C). p-STAT1 and STAT1 were detected by Western blot with β-actin used as a loading control. (D). Glutamate was detected in supernatants by HPLC. (E). Intracellular glutamate was detected using the Amplex ® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data (A-E) are representative of three independent experiments using three different donors. (F) Human MDM were infected with HIV-1 for 5 days, then total RNA was collected. IFN-α and IFN-β mRNA levels were determined by real-time RT-PCR. Results shown are representative of three independent experiments using three different donors and are means of triplicate samples. (G-I) Human MDM were infected with HIV-1 for 5 days in the presence of IgG or IFN-α/IFN-β neutralizing antibodies. Total RNA and cell lysates were collected and GAC mRNA (G) and protein (H and I) levels were determined by real time RT-PCR and Western blot, respectively. For quantification, GAC expression were normalized as a ratio to β-actin and shown as fold change relative to IgG control . Results (F, I) are shown as the average ± SEM of three independent experiments with three different donors, #, p

    Techniques Used: Infection, Chromatin Immunoprecipitation, Negative Control, Activity Assay, Western Blot, High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing

    STAT1 phosphorylation is required for IFN-α to activate the GLS1 promoter and induce glutaminase expression and function. (A). MDM were treated with different doses of IFN-α for 24 hours, then p-STAT1 (Tyr 701), STAT1, KGA and GAC were detected by Western blot. β-actin was used as a loading control. (B). Intracellular glutamate was detected using the Amplex ® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data are representative of three independent experiments using three different donors and are the means of triplicate samples. (C-F). Levels of p-STAT1 (C), STAT1 (D), GAC (E) and KGA (F) in Western blot (A) were normalized as a ratio to β-actin and shown as fold change relative to control. Results are shown as the average ± SEM of three independent experiments with three different donors, (G, H). Correlation of p-STAT1 with GAC (G) and p-STAT1 with KGA (H) in representative donor are shown. (I, J). HEK 293T cells were co-transfected with the GLS1 promoter construct and pRL-SV40. 24 hours later, the cells were pretreated with 1 µM fludarabine or 1:10,000 DMSO for 1 hour, then treated with or without 100 U/ml IFN-α for 24 hours. (I). p-STAT1 and STAT1 were detected by Western blot, β-actin was used as a loading control. (J). Luciferase activity in the lysates was measured by luminescence detection. Renilla luciferase was used to normalize transfection efficiency. The data are representative of three independent experiments and are the means of triplicate samples. #, p
    Figure Legend Snippet: STAT1 phosphorylation is required for IFN-α to activate the GLS1 promoter and induce glutaminase expression and function. (A). MDM were treated with different doses of IFN-α for 24 hours, then p-STAT1 (Tyr 701), STAT1, KGA and GAC were detected by Western blot. β-actin was used as a loading control. (B). Intracellular glutamate was detected using the Amplex ® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data are representative of three independent experiments using three different donors and are the means of triplicate samples. (C-F). Levels of p-STAT1 (C), STAT1 (D), GAC (E) and KGA (F) in Western blot (A) were normalized as a ratio to β-actin and shown as fold change relative to control. Results are shown as the average ± SEM of three independent experiments with three different donors, (G, H). Correlation of p-STAT1 with GAC (G) and p-STAT1 with KGA (H) in representative donor are shown. (I, J). HEK 293T cells were co-transfected with the GLS1 promoter construct and pRL-SV40. 24 hours later, the cells were pretreated with 1 µM fludarabine or 1:10,000 DMSO for 1 hour, then treated with or without 100 U/ml IFN-α for 24 hours. (I). p-STAT1 and STAT1 were detected by Western blot, β-actin was used as a loading control. (J). Luciferase activity in the lysates was measured by luminescence detection. Renilla luciferase was used to normalize transfection efficiency. The data are representative of three independent experiments and are the means of triplicate samples. #, p

    Techniques Used: Expressing, Western Blot, Transfection, Construct, Luciferase, Activity Assay

    Significant correlation between STAT1 and GAC mRNA levels in brain tissues of HAD patients. Total RNA from post-mortem brain tissue collected from HAD patients, HIV serum-positive patients without dementia, and HIV serum negative individuals were extracted and subjected to real time RT-PCR for STAT1, GAC, IFN-α2 and IFN-β. GAPDH expression was used as an internal control. (A) Levels of STAT1 are normalized as a ratio to GAPDH and shown as fold change relative to the average of HIV serum negative controls. Results are shown as the average ± SEM, #, p
    Figure Legend Snippet: Significant correlation between STAT1 and GAC mRNA levels in brain tissues of HAD patients. Total RNA from post-mortem brain tissue collected from HAD patients, HIV serum-positive patients without dementia, and HIV serum negative individuals were extracted and subjected to real time RT-PCR for STAT1, GAC, IFN-α2 and IFN-β. GAPDH expression was used as an internal control. (A) Levels of STAT1 are normalized as a ratio to GAPDH and shown as fold change relative to the average of HIV serum negative controls. Results are shown as the average ± SEM, #, p

    Techniques Used: Quantitative RT-PCR, Expressing

    15) Product Images from "Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases"

    Article Title: Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-12-1645

    IL-12Rβ1 plays a central role in IL-12 signal transduction. (A) Selected IL‑12Rβ1 variants were retrovirally transduced in Ba/F3-gp130-IL‑12Rβ2 cells. Representative histograms of IL‑12Rβ1 (top) and IL‑12Rβ2 (bottom) surface expression of resulting transduced Ba/F3-gp130 cell lines. Gray-shaded areas indicate Ba/F3-gp130 cells (negative control), and dark solid lines are the respective Ba/F3 cell lines as indicated. (B) Proliferation of stably transduced Ba/F3-gp130-IL-12Rβ2 cells with cDNAs coding for murine IL‑12Rβ1 (WT) or appropriate variants. Equal numbers of cells were cultured for 3 d in the presence of 0.2% HIL‑6–conditioned cell culture supernatant or 0.4% HIL‑12 or without cytokine. Parental Ba/F3-gp130, Ba/F3-gp130-mIL-12Rβ1, and Ba/F3-gp130-mIL‑12Rβ2 cells were used as control. Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL‑6–dependent proliferation was set to 100%. Error bars represent SD for technical replicates. (C) Stably transduced Ba/F3 cells were washed three times, starved, and stimulated with 0.2% HIL‑6 or 0.4% HIL‑12 for 15 or 30 min. Cellular lysates were prepared, and 50 μg of total protein per lane was loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-STAT1, STAT1, phospho-Erk1/2, and Erk1/2. Ba/F3-gp130-mIL‑12Rβ1 and Ba/F3-gp130-mIL-12Rβ2 cells were used as control. Western blot data show one representative experiment.
    Figure Legend Snippet: IL-12Rβ1 plays a central role in IL-12 signal transduction. (A) Selected IL‑12Rβ1 variants were retrovirally transduced in Ba/F3-gp130-IL‑12Rβ2 cells. Representative histograms of IL‑12Rβ1 (top) and IL‑12Rβ2 (bottom) surface expression of resulting transduced Ba/F3-gp130 cell lines. Gray-shaded areas indicate Ba/F3-gp130 cells (negative control), and dark solid lines are the respective Ba/F3 cell lines as indicated. (B) Proliferation of stably transduced Ba/F3-gp130-IL-12Rβ2 cells with cDNAs coding for murine IL‑12Rβ1 (WT) or appropriate variants. Equal numbers of cells were cultured for 3 d in the presence of 0.2% HIL‑6–conditioned cell culture supernatant or 0.4% HIL‑12 or without cytokine. Parental Ba/F3-gp130, Ba/F3-gp130-mIL-12Rβ1, and Ba/F3-gp130-mIL‑12Rβ2 cells were used as control. Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL‑6–dependent proliferation was set to 100%. Error bars represent SD for technical replicates. (C) Stably transduced Ba/F3 cells were washed three times, starved, and stimulated with 0.2% HIL‑6 or 0.4% HIL‑12 for 15 or 30 min. Cellular lysates were prepared, and 50 μg of total protein per lane was loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-STAT1, STAT1, phospho-Erk1/2, and Erk1/2. Ba/F3-gp130-mIL‑12Rβ1 and Ba/F3-gp130-mIL-12Rβ2 cells were used as control. Western blot data show one representative experiment.

    Techniques Used: Transduction, Expressing, Negative Control, Stable Transfection, Cell Culture, Viability Assay, Western Blot

    16) Product Images from "Hepatitis-C-virus-induced microRNAs dampen interferon-mediated antiviral signaling"

    Article Title: Hepatitis-C-virus-induced microRNAs dampen interferon-mediated antiviral signaling

    Journal: Nature medicine

    doi: 10.1038/nm.4211

    myomiR overexpression reduces IFNAR1 expression, dampening downstream Jak-STAT signaling and induction of ISGs. ( a ) IFNAR1 and IFNAR2 mRNA expression in the hepatocyte cell lines HepG2, PH5CH8, or Huh7 transfected with mimics for miR-208b, miR-499a-5p, a mixture of both mimics (myomiRs) or a control mimic (NC). Reference sample for relative expression was NC-transfected cells. ns, non-significant. ( b ) IFNAR1 surface protein expression in HepG2 cells stably overexpressing miR-208b (pLV-208b) or miR-499a-5p (pLV-499a-5p). ( c ) Stability of IFNAR1 and IFNAR2 mRNA in HepG2 cells transfected with myomiR mimics or NC mimic and treated with actinomycin D to arrest new transcription, presented as mRNA remaining over time relative to that at 0 h, set as 100%. R 2 ≥ 0.63 for all curve fits. Half-lives (50% mRNA remaining): IFNAR1 , 34.5 h (NC) and 14.1 h (myomiR); IFNAR2 , 9.9 h (NC) and 10.5 h (myomiR). * P = 0.0003 ( F test). ( d ) Immunoblot of lysates from HepG2 cells transfected with myomiR mimics or NC mimic and stimulated with 500 IU/mL IFN-β for 0, 15, 30, or 60 min. Blot was probed for STAT1, STAT2, Jak1, Tyk2 and their phosphorylated forms. β-actin was run as a loading control. ( e ) Expression of ISGs MX1 , OAS1 and PKR in HepG2 cells transfected with myomiR mimics or NC mimic stimulated with 500 IU/mL IFN-β for 6 h. Relative quantification was computed using the respective unstimulated cells then normalized to NC fold change and expressed as % mRNA of NC. Data are from ( a ) one experiment of four experiments with similar results, ( b ) one experiment representative of two experiments with four replicates per group or ( c – e ) one experiment of two experiments with similar results ( a , c , e , mean ± s.e.m.; b , c , g , box and whisker plot shows median values (line) and minimum and maximum values (whiskers)). One-way ANOVA ( a ) or unpaired Student’s t -test ( b , c , e ) was used for statistical comparison, * P
    Figure Legend Snippet: myomiR overexpression reduces IFNAR1 expression, dampening downstream Jak-STAT signaling and induction of ISGs. ( a ) IFNAR1 and IFNAR2 mRNA expression in the hepatocyte cell lines HepG2, PH5CH8, or Huh7 transfected with mimics for miR-208b, miR-499a-5p, a mixture of both mimics (myomiRs) or a control mimic (NC). Reference sample for relative expression was NC-transfected cells. ns, non-significant. ( b ) IFNAR1 surface protein expression in HepG2 cells stably overexpressing miR-208b (pLV-208b) or miR-499a-5p (pLV-499a-5p). ( c ) Stability of IFNAR1 and IFNAR2 mRNA in HepG2 cells transfected with myomiR mimics or NC mimic and treated with actinomycin D to arrest new transcription, presented as mRNA remaining over time relative to that at 0 h, set as 100%. R 2 ≥ 0.63 for all curve fits. Half-lives (50% mRNA remaining): IFNAR1 , 34.5 h (NC) and 14.1 h (myomiR); IFNAR2 , 9.9 h (NC) and 10.5 h (myomiR). * P = 0.0003 ( F test). ( d ) Immunoblot of lysates from HepG2 cells transfected with myomiR mimics or NC mimic and stimulated with 500 IU/mL IFN-β for 0, 15, 30, or 60 min. Blot was probed for STAT1, STAT2, Jak1, Tyk2 and their phosphorylated forms. β-actin was run as a loading control. ( e ) Expression of ISGs MX1 , OAS1 and PKR in HepG2 cells transfected with myomiR mimics or NC mimic stimulated with 500 IU/mL IFN-β for 6 h. Relative quantification was computed using the respective unstimulated cells then normalized to NC fold change and expressed as % mRNA of NC. Data are from ( a ) one experiment of four experiments with similar results, ( b ) one experiment representative of two experiments with four replicates per group or ( c – e ) one experiment of two experiments with similar results ( a , c , e , mean ± s.e.m.; b , c , g , box and whisker plot shows median values (line) and minimum and maximum values (whiskers)). One-way ANOVA ( a ) or unpaired Student’s t -test ( b , c , e ) was used for statistical comparison, * P

    Techniques Used: Over Expression, Expressing, Transfection, Stable Transfection, Whisker Assay

    17) Product Images from "Suppressor of Cytokine Signaling Expression with Increasing Severity of Murine Hepatic Ischemia-Reperfusion Injury"

    Article Title: Suppressor of Cytokine Signaling Expression with Increasing Severity of Murine Hepatic Ischemia-Reperfusion Injury

    Journal:

    doi: 10.1016/j.jhep.2008.03.014

    Activation of STAT1 and STAT3 after Hepatic IR
    Figure Legend Snippet: Activation of STAT1 and STAT3 after Hepatic IR

    Techniques Used: Activation Assay

    18) Product Images from "A novel 1,2-benzenediamine derivative FC-99 suppresses TLR3 expression and ameliorates disease symptoms in a mouse model of sepsis"

    Article Title: A novel 1,2-benzenediamine derivative FC-99 suppresses TLR3 expression and ameliorates disease symptoms in a mouse model of sepsis

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12797

    FC-99 reduced the TLR3 expression induced by exogenous IFN-α via STAT1. RAW264.7 cells (A) or peritoneal macrophage (B) was pretreated with FC-99 for 2 h before IFN-α (100 U·mL −1 ) stimulation for another 3 h and TLR3 mRNA levels were tested by Q-PCR ( n = 4). (C, D) RAW264.7 cells were treated with IFN-α (100 U·mL −1 ) for the indicated time with or without pretreatment with FC-99 for 2 h; protein levels of phospho-STAT1, STAT1 phospho-JAK1, JAK1 and GAPDH were tested by Western blot. Histograms show the relative band intensity from three independent experiments. The data are means ± SEM. *** P
    Figure Legend Snippet: FC-99 reduced the TLR3 expression induced by exogenous IFN-α via STAT1. RAW264.7 cells (A) or peritoneal macrophage (B) was pretreated with FC-99 for 2 h before IFN-α (100 U·mL −1 ) stimulation for another 3 h and TLR3 mRNA levels were tested by Q-PCR ( n = 4). (C, D) RAW264.7 cells were treated with IFN-α (100 U·mL −1 ) for the indicated time with or without pretreatment with FC-99 for 2 h; protein levels of phospho-STAT1, STAT1 phospho-JAK1, JAK1 and GAPDH were tested by Western blot. Histograms show the relative band intensity from three independent experiments. The data are means ± SEM. *** P

    Techniques Used: Expressing, Polymerase Chain Reaction, Western Blot

    19) Product Images from "Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia"

    Article Title: Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070352

    Differential expression of CTLA4 and associated molecules in primary CLL cells. Panel I: Differential expression of CTLA4 and associated molecules in high-CD38/low-CTLA4 and low-CD38/high-CTLA4 CLL subgroups as determined by microarray data. Subpanel A: Mean normalized expression levels of CTLA4 in the CD38-low group compared to the CD38-high group. Subpanels B–D: Mean normalized expression levels of STAT1, NFATC2, and c-Fos (respectively) in the CTLA4-high group compared to CTLA4-low group. Panel II: Overexpression of downstream molecules in BCR proliferation signaling in two prognostic CLL subgroups, as determined by real-time PCR. Subpanels A–C: Real-time PCR showing gene expression for STAT1, NFATC2, and c-Fos (fold change) in the high-CTLA4 CLL group compared to the low-CTLA4 CLL group, respectively. Subpanels D–F: Real-time PCR showing gene expression of STAT1, NFATC2, and c-Fos (fold change) in chromosomal abnormality subgroups, respectively (good outcome includes 13q14 deletion and normal karyotype, while poor outcome includes trisomy12, 11q deletion, and 17p deletion chromosomal abnormality).
    Figure Legend Snippet: Differential expression of CTLA4 and associated molecules in primary CLL cells. Panel I: Differential expression of CTLA4 and associated molecules in high-CD38/low-CTLA4 and low-CD38/high-CTLA4 CLL subgroups as determined by microarray data. Subpanel A: Mean normalized expression levels of CTLA4 in the CD38-low group compared to the CD38-high group. Subpanels B–D: Mean normalized expression levels of STAT1, NFATC2, and c-Fos (respectively) in the CTLA4-high group compared to CTLA4-low group. Panel II: Overexpression of downstream molecules in BCR proliferation signaling in two prognostic CLL subgroups, as determined by real-time PCR. Subpanels A–C: Real-time PCR showing gene expression for STAT1, NFATC2, and c-Fos (fold change) in the high-CTLA4 CLL group compared to the low-CTLA4 CLL group, respectively. Subpanels D–F: Real-time PCR showing gene expression of STAT1, NFATC2, and c-Fos (fold change) in chromosomal abnormality subgroups, respectively (good outcome includes 13q14 deletion and normal karyotype, while poor outcome includes trisomy12, 11q deletion, and 17p deletion chromosomal abnormality).

    Techniques Used: Expressing, Microarray, Over Expression, Real-time Polymerase Chain Reaction

    Upregulation of STAT1/phospho-STAT1, NFATC2, c-Fos/phospho-c-Fos, and c-Myc in CTLA4-downregulated CLL (low CD38/high CTLA4) cells as determined by RT-PCR, real-time PCR, and/or Western blotting. Panels A: Semi-quantitative RT-PCR showing that downregulation of CTLA4 by AS in CLL cells leads to up-regulation of STAT1, NFATC2, c-Fos, and c-Myc in CLL. Panel B: Real-time PCR results from four patient samples showing a significantly higher expression of c-Myc in CTLA4-downregulated CLL cells compared to control CLL cells or CLL cells treated with irrelevant AS. Panel C–D: Western blot results showing up-regulation and quantification of the expression of STAT1 and its phosphorylation, c-Fos and its phosphorylation, NFATC2, and c-Myc at the protein level in CTLA4 down-regulated CLL patient samples (n = 3). *p indicates the statistical difference between control CLL cells and CTLA4-downregulated CLL cells. β-Actin was used as a control.
    Figure Legend Snippet: Upregulation of STAT1/phospho-STAT1, NFATC2, c-Fos/phospho-c-Fos, and c-Myc in CTLA4-downregulated CLL (low CD38/high CTLA4) cells as determined by RT-PCR, real-time PCR, and/or Western blotting. Panels A: Semi-quantitative RT-PCR showing that downregulation of CTLA4 by AS in CLL cells leads to up-regulation of STAT1, NFATC2, c-Fos, and c-Myc in CLL. Panel B: Real-time PCR results from four patient samples showing a significantly higher expression of c-Myc in CTLA4-downregulated CLL cells compared to control CLL cells or CLL cells treated with irrelevant AS. Panel C–D: Western blot results showing up-regulation and quantification of the expression of STAT1 and its phosphorylation, c-Fos and its phosphorylation, NFATC2, and c-Myc at the protein level in CTLA4 down-regulated CLL patient samples (n = 3). *p indicates the statistical difference between control CLL cells and CTLA4-downregulated CLL cells. β-Actin was used as a control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Expressing

    Hypothetical model for the role of CTLA4 in CLL cell proliferation/survival. Panel A: When CLL cells express low CD38, but high CTLA4, CTLA4 inhibits the CD38/BCR signaling pathway at multiple levels. CTLA4 downregulates NFATC2 and proliferation-associated molecules such as c-Fos and c-Myc. Downregulation of NFATC2 may also be associated with an autoregulatory loop for CTLA4, which would downregulate CTLA4 transcription. CTLA4 also downregulates the expression of Bcl-2, thus decreasing the survival of CLL cells. CTLA4 inhibits the expression of STAT1, thus deregulating the JAK/STAT pathway and inhibiting CLL cell growth. Panel B: When CLL cells express high CD38, but low CTLA4, activated CD38/BCR signaling upregulates downstream molecules in the pathway, such as NFATC2, c-Fos, and Bcl-2. These molecules will increase proliferation and survival of CLL cells. Low expression of CTLA4 does not interfere with the expression of STAT1, which favors CLL cell growth.
    Figure Legend Snippet: Hypothetical model for the role of CTLA4 in CLL cell proliferation/survival. Panel A: When CLL cells express low CD38, but high CTLA4, CTLA4 inhibits the CD38/BCR signaling pathway at multiple levels. CTLA4 downregulates NFATC2 and proliferation-associated molecules such as c-Fos and c-Myc. Downregulation of NFATC2 may also be associated with an autoregulatory loop for CTLA4, which would downregulate CTLA4 transcription. CTLA4 also downregulates the expression of Bcl-2, thus decreasing the survival of CLL cells. CTLA4 inhibits the expression of STAT1, thus deregulating the JAK/STAT pathway and inhibiting CLL cell growth. Panel B: When CLL cells express high CD38, but low CTLA4, activated CD38/BCR signaling upregulates downstream molecules in the pathway, such as NFATC2, c-Fos, and Bcl-2. These molecules will increase proliferation and survival of CLL cells. Low expression of CTLA4 does not interfere with the expression of STAT1, which favors CLL cell growth.

    Techniques Used: Expressing

    20) Product Images from "Ubiquitin-activating enzyme E1 inhibitor PYR41 attenuates angiotensin II-induced activation of dendritic cells via the IκBa/NF-κB and MKP1/ERK/STAT1 pathways"

    Article Title: Ubiquitin-activating enzyme E1 inhibitor PYR41 attenuates angiotensin II-induced activation of dendritic cells via the IκBa/NF-κB and MKP1/ERK/STAT1 pathways

    Journal: Immunology

    doi: 10.1111/imm.12255

    PYR41 suppresses the activation of the mitogen-activated protein kinase phosphatase 1 (MKP-1)/ extracellular signal-regulated kinase (ERK)/ signal transducer and activator of transcription 1 (STAT1) pathway in dendritic cells (DCs) induced by Angiotensin
    Figure Legend Snippet: PYR41 suppresses the activation of the mitogen-activated protein kinase phosphatase 1 (MKP-1)/ extracellular signal-regulated kinase (ERK)/ signal transducer and activator of transcription 1 (STAT1) pathway in dendritic cells (DCs) induced by Angiotensin

    Techniques Used: Activation Assay

    21) Product Images from "Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages"

    Article Title: Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1121131109

    Impaired Ifn-β and Stat1 expression in Hdac3 −/− macrophages. ( A and B ) Western blots were hybridized with the indicated antibodies. Vinculin was used as a loading control. Stat1 ( C ) and Ifnb ( D ) mRNA expression was measured by
    Figure Legend Snippet: Impaired Ifn-β and Stat1 expression in Hdac3 −/− macrophages. ( A and B ) Western blots were hybridized with the indicated antibodies. Vinculin was used as a loading control. Stat1 ( C ) and Ifnb ( D ) mRNA expression was measured by

    Techniques Used: Expressing, Western Blot

    22) Product Images from "Changes in Susceptibility to Oncolytic Vesicular Stomatitis Virus during Progression of Prostate Cancer"

    Article Title: Changes in Susceptibility to Oncolytic Vesicular Stomatitis Virus during Progression of Prostate Cancer

    Journal: Journal of Virology

    doi: 10.1128/JVI.00257-15

    Tumor-derived Pten −/− cells constitutively express high levels of STAT1, which is phosphorylated in response to infection with VSV with either wt or mutant M protein. Control Pten L/L , acutely deleted, and tumor-derived Pten −/−
    Figure Legend Snippet: Tumor-derived Pten −/− cells constitutively express high levels of STAT1, which is phosphorylated in response to infection with VSV with either wt or mutant M protein. Control Pten L/L , acutely deleted, and tumor-derived Pten −/−

    Techniques Used: Derivative Assay, Infection, Mutagenesis

    23) Product Images from "Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma"

    Article Title: Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181885

    LY5 inhibits pSTAT3 in sarcoma cell lines. (A) The tumor cell lines RH30, EW8, and RD were treated with increasing concentrations of LY5 for 30 minutes. Cell lysates were collected for Western blotting of p-STAT3, total STAT3, and GAPDH. GAPDH was used as loading control. (B-C) The SJSA cell line was serum-starved overnight and then left untreated or treated with LY5 for two hours followed by stimulation with 50 ng/mL of OSM, IL-6, IFN-γ, IFN-α, or IL-4 for 30 minutes prior to collection of cell lysates for Western blotting of p-STAT3, total STAT3 p-STAT1, total STAT1, p-STAT2, p-STAT4, or p-STAT6. GAPDH was used as loading control.
    Figure Legend Snippet: LY5 inhibits pSTAT3 in sarcoma cell lines. (A) The tumor cell lines RH30, EW8, and RD were treated with increasing concentrations of LY5 for 30 minutes. Cell lysates were collected for Western blotting of p-STAT3, total STAT3, and GAPDH. GAPDH was used as loading control. (B-C) The SJSA cell line was serum-starved overnight and then left untreated or treated with LY5 for two hours followed by stimulation with 50 ng/mL of OSM, IL-6, IFN-γ, IFN-α, or IL-4 for 30 minutes prior to collection of cell lysates for Western blotting of p-STAT3, total STAT3 p-STAT1, total STAT1, p-STAT2, p-STAT4, or p-STAT6. GAPDH was used as loading control.

    Techniques Used: Western Blot

    24) Product Images from "Type I Interferons and Interferon Regulatory Factors Regulate TNF-Related Apoptosis-Inducing Ligand (TRAIL) in HIV-1-Infected Macrophages"

    Article Title: Type I Interferons and Interferon Regulatory Factors Regulate TNF-Related Apoptosis-Inducing Ligand (TRAIL) in HIV-1-Infected Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005397

    Fludarabine blocks HIV-1-induced STAT1 activation and gene expression of IRF-1, IRF-7, and TRAIL in macrophages. A. MDM were treated with fludarabine at 1 µM 3 days after infection. P-STAT1 and total STAT1 were detected by Western blotting at 5 days after infection. β-actin was used as a loading control. B–D. Real-time RT-PCR was used to detect TRAIL (B), IRF-1 (C), and IRF-7 (D). **, p
    Figure Legend Snippet: Fludarabine blocks HIV-1-induced STAT1 activation and gene expression of IRF-1, IRF-7, and TRAIL in macrophages. A. MDM were treated with fludarabine at 1 µM 3 days after infection. P-STAT1 and total STAT1 were detected by Western blotting at 5 days after infection. β-actin was used as a loading control. B–D. Real-time RT-PCR was used to detect TRAIL (B), IRF-1 (C), and IRF-7 (D). **, p

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

    HIV-1 infection induces IRF-1 and IRF-7 gene expression and STAT1 phosphorylation at Tyr701 in macrophages. MDM were infected with HIV-1 and cell lysates and RNA were collected 1, 3, 5, and 7 days after infection. A–C. Real-time RT-PCR was used to detect IRF-1 (A), IRF-3 (B), and IRF-7 (C). Open bars represent control MDM and solid bars represent HIV-1-infected MDM. IFN-α (1000 Units/ml) was also used to stimulate MDM for 24 hours, the effect on IRFs expression is shown in each panel as the diagonal striped bar. D. Phospho-STAT1 (p-STAT1, Tyr701) and total STAT1 were detected by Western blotting. β-actin was used as a loading control. E. Levels of p-STAT1 were normalized as a ratio of p-STAT1 to STAT1 after densimetrical quantification of panel D and shown as fold change relative to control (1 dpi). F. Levels of STAT1 were normalized as a ratio of STAT1 to β-actin and shown as fold change relative to control (1 dpi). Results are shown as the average±SEM in experiments performed with three different donors. *, p
    Figure Legend Snippet: HIV-1 infection induces IRF-1 and IRF-7 gene expression and STAT1 phosphorylation at Tyr701 in macrophages. MDM were infected with HIV-1 and cell lysates and RNA were collected 1, 3, 5, and 7 days after infection. A–C. Real-time RT-PCR was used to detect IRF-1 (A), IRF-3 (B), and IRF-7 (C). Open bars represent control MDM and solid bars represent HIV-1-infected MDM. IFN-α (1000 Units/ml) was also used to stimulate MDM for 24 hours, the effect on IRFs expression is shown in each panel as the diagonal striped bar. D. Phospho-STAT1 (p-STAT1, Tyr701) and total STAT1 were detected by Western blotting. β-actin was used as a loading control. E. Levels of p-STAT1 were normalized as a ratio of p-STAT1 to STAT1 after densimetrical quantification of panel D and shown as fold change relative to control (1 dpi). F. Levels of STAT1 were normalized as a ratio of STAT1 to β-actin and shown as fold change relative to control (1 dpi). Results are shown as the average±SEM in experiments performed with three different donors. *, p

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot

    siRNA knockdown of IRF-1 and IRF-7 reduces STAT1 phosphorylation and TRAIL expression in HIV-1-infected macrophages. Two days after HIV-1 infection, MDM were transfected with siRNA for IRF-1, -3, or -7. A. Forty-eight hours later, successful transfections were confirmed by Silencer FAM-labeled Negative Control #1 siRNA transfection indicator (green). Hoechst 33258 (nucleus marker, blue) was used to visualize the total cell number. B–D. Total RNA was collected 48 hours post-transfection and mRNA levels of IRF-1(B), -3(C), or -7(D) were determined by real-time RT-PCR. E. Ninety-six hours after transfection, p-STAT1 and total STAT1 were detected by Western blotting. β-actin was used as a loading control. F. TRAIL expression levels were determined by real-time RT-PCR. Results were normalized with GAPDH and shown as the fold change over non-specific siRNA control. G. Supernatants were tested for HIV-1 RTase activity. ** indicates p
    Figure Legend Snippet: siRNA knockdown of IRF-1 and IRF-7 reduces STAT1 phosphorylation and TRAIL expression in HIV-1-infected macrophages. Two days after HIV-1 infection, MDM were transfected with siRNA for IRF-1, -3, or -7. A. Forty-eight hours later, successful transfections were confirmed by Silencer FAM-labeled Negative Control #1 siRNA transfection indicator (green). Hoechst 33258 (nucleus marker, blue) was used to visualize the total cell number. B–D. Total RNA was collected 48 hours post-transfection and mRNA levels of IRF-1(B), -3(C), or -7(D) were determined by real-time RT-PCR. E. Ninety-six hours after transfection, p-STAT1 and total STAT1 were detected by Western blotting. β-actin was used as a loading control. F. TRAIL expression levels were determined by real-time RT-PCR. Results were normalized with GAPDH and shown as the fold change over non-specific siRNA control. G. Supernatants were tested for HIV-1 RTase activity. ** indicates p

    Techniques Used: Expressing, Infection, Transfection, Labeling, Negative Control, Marker, Quantitative RT-PCR, Western Blot, Activity Assay

    A feedback loop among Type I IFNs and IRFs regulates TRAIL Expression in HIV-1-infected macrophages. A positive feedback loop exists among intracellular IRFs gene expression and soluble type I IFNs induction in macrophages during HIV-1 infection. HIV-1 infection induces IRF-1 and IRF-7 gene expression. IRF-1 and IRF-7 promotes type I IFNs activities and activate STAT1. Type I IFNs diffuse and further promote STAT1 activation and IRF-1 and IRF-7 expression in uninfected macrophages. Blocking of either IRF-1, IRF-7, type I IFNs, or STAT1 reduces TRAIL expression. TRAIL has been reported to mediate the apoptosis of CD4+ T cells and participate in HIV-1 pathogenesis.
    Figure Legend Snippet: A feedback loop among Type I IFNs and IRFs regulates TRAIL Expression in HIV-1-infected macrophages. A positive feedback loop exists among intracellular IRFs gene expression and soluble type I IFNs induction in macrophages during HIV-1 infection. HIV-1 infection induces IRF-1 and IRF-7 gene expression. IRF-1 and IRF-7 promotes type I IFNs activities and activate STAT1. Type I IFNs diffuse and further promote STAT1 activation and IRF-1 and IRF-7 expression in uninfected macrophages. Blocking of either IRF-1, IRF-7, type I IFNs, or STAT1 reduces TRAIL expression. TRAIL has been reported to mediate the apoptosis of CD4+ T cells and participate in HIV-1 pathogenesis.

    Techniques Used: Expressing, Infection, Activation Assay, Blocking Assay

    Type I interferon from HIV-1-infected macrophages induces STAT1 phosphorylation, IRF-1, IRF-7, and TRAIL expression. A. MDM were treated with IFN-α (1000 Units/ml) or IFN-β (1000 Units/ml) with or without their corresponding neutralizing antibodies. Cell lysates were collected 2 hours later and subjected to Western blotting for p-STAT1 and STAT1. β-actin was used as a loading control. B. 24 hours after the treatment, TRAIL expression was determined by real-time RT-PCR. ** denotes p
    Figure Legend Snippet: Type I interferon from HIV-1-infected macrophages induces STAT1 phosphorylation, IRF-1, IRF-7, and TRAIL expression. A. MDM were treated with IFN-α (1000 Units/ml) or IFN-β (1000 Units/ml) with or without their corresponding neutralizing antibodies. Cell lysates were collected 2 hours later and subjected to Western blotting for p-STAT1 and STAT1. β-actin was used as a loading control. B. 24 hours after the treatment, TRAIL expression was determined by real-time RT-PCR. ** denotes p

    Techniques Used: Infection, Expressing, Western Blot, Quantitative RT-PCR

    25) Product Images from "Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism"

    Article Title: Regulation of iNOS Gene Transcription by IL-1β and IFN-γ Requires a Coactivator Exchange Mechanism

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2013-1159

    Phosphorylation of STAT1 at Ser727 is critical for IL-1β-stimulated transcription of the iNOS gene. A, 832/13 cells were cotransfected with WT or cGAS −1-kb constructs and either an siRNA duplex containing a control sequence (siScramble)
    Figure Legend Snippet: Phosphorylation of STAT1 at Ser727 is critical for IL-1β-stimulated transcription of the iNOS gene. A, 832/13 cells were cotransfected with WT or cGAS −1-kb constructs and either an siRNA duplex containing a control sequence (siScramble)

    Techniques Used: Construct, Sequencing

    26) Product Images from "The CPLANE protein Intu protects kidneys from ischemia-reperfusion injury by targeting STAT1 for degradation"

    Article Title: The CPLANE protein Intu protects kidneys from ischemia-reperfusion injury by targeting STAT1 for degradation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03628-8

    Intu targets STAT1 for degradation. a Co-transfection of Intu-mycDDK reduced the expression of FLAG-tagged STAT1α and its phosphorylation mutants (STAT1αY701F, STAT1αS727A) in 293FT cells. b Intu suppressed the expression of total and phosphorylated STAT1 during IFN-γ, azide, or cisplatin treatment of 293FT cells. c Intu or its deletion mutants were co-transfected with STAT1-FLAG into 293FT cells, showing the suppressive effect of full-length Intu and Intu ∆268-942 on STAT1 expression. d MG132 improved STAT1 expression in Intu-transfected cells. e Higher levels of total and phosphorylated STAT1, cleaved Caspase-3, and Parp, and lower levels of Bcl-2 and Bcl-xL were observed in PT-Intu-KO kidney tissues, in comparison to the wild type. Quantification was done by densitometry of immunoblots ( n = 3–4). Quantitative data are mean ± s.d. (error bars). Paired t test was used. * p
    Figure Legend Snippet: Intu targets STAT1 for degradation. a Co-transfection of Intu-mycDDK reduced the expression of FLAG-tagged STAT1α and its phosphorylation mutants (STAT1αY701F, STAT1αS727A) in 293FT cells. b Intu suppressed the expression of total and phosphorylated STAT1 during IFN-γ, azide, or cisplatin treatment of 293FT cells. c Intu or its deletion mutants were co-transfected with STAT1-FLAG into 293FT cells, showing the suppressive effect of full-length Intu and Intu ∆268-942 on STAT1 expression. d MG132 improved STAT1 expression in Intu-transfected cells. e Higher levels of total and phosphorylated STAT1, cleaved Caspase-3, and Parp, and lower levels of Bcl-2 and Bcl-xL were observed in PT-Intu-KO kidney tissues, in comparison to the wild type. Quantification was done by densitometry of immunoblots ( n = 3–4). Quantitative data are mean ± s.d. (error bars). Paired t test was used. * p

    Techniques Used: Cotransfection, Expressing, Transfection, Western Blot

    Schematic model of Intu/STAT1 regulation of cell death and cilium in kidney injury. In wild-type cells and tissues, renal IRI upregulates Intu, which binds and sequesters STAT1 in the basal body/centriole area for degradation, reducing phosphorylated STAT1 translocation to the nucleus and alleviating cell death (upper). Intu-KO cells and tissues are devoid of Intu, the STAT1 antagonizing mechanism, and as a result, more phosphorylated STAT1 accumulates in the nucleus to promote cell death (lower). Also, Intu promotes ciliogenesis, whereas STAT1 may have opposite effects. Phosphorylated STAT1 is marked with a circled P in red for Y701 and in black for S727
    Figure Legend Snippet: Schematic model of Intu/STAT1 regulation of cell death and cilium in kidney injury. In wild-type cells and tissues, renal IRI upregulates Intu, which binds and sequesters STAT1 in the basal body/centriole area for degradation, reducing phosphorylated STAT1 translocation to the nucleus and alleviating cell death (upper). Intu-KO cells and tissues are devoid of Intu, the STAT1 antagonizing mechanism, and as a result, more phosphorylated STAT1 accumulates in the nucleus to promote cell death (lower). Also, Intu promotes ciliogenesis, whereas STAT1 may have opposite effects. Phosphorylated STAT1 is marked with a circled P in red for Y701 and in black for S727

    Techniques Used: Translocation Assay

    Intu protects cells from death through STAT1. a Inducible expression of Intu suppressed STAT1 expression and Caspase-3 cleavage during azide and cisplatin treatment. b Inducible expression of Intu suppressed azide-induced cell death in STAT1α-transfected cells ( n = 5). c After exposure to cisplatin, Intu/STAT1 double overexpression cells showed resistance as compared to STAT1 single overexpression cells ( n = 8). d Confirmation of STAT1 knockdown by shRNAs in Intu/STAT1 double knockdown cells. e Knockdown of STAT1 inhibited azide-induced Caspase-3 cleavage/activation in Intu-silenced cells. f Knockdown of STAT1 rescued Intu-silenced cells from azide-induced cell death ( n = 12). g Knockdown of STAT1 rescued Intu-silenced cells from cisplatin-induced cell death ( n = 12). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p
    Figure Legend Snippet: Intu protects cells from death through STAT1. a Inducible expression of Intu suppressed STAT1 expression and Caspase-3 cleavage during azide and cisplatin treatment. b Inducible expression of Intu suppressed azide-induced cell death in STAT1α-transfected cells ( n = 5). c After exposure to cisplatin, Intu/STAT1 double overexpression cells showed resistance as compared to STAT1 single overexpression cells ( n = 8). d Confirmation of STAT1 knockdown by shRNAs in Intu/STAT1 double knockdown cells. e Knockdown of STAT1 inhibited azide-induced Caspase-3 cleavage/activation in Intu-silenced cells. f Knockdown of STAT1 rescued Intu-silenced cells from azide-induced cell death ( n = 12). g Knockdown of STAT1 rescued Intu-silenced cells from cisplatin-induced cell death ( n = 12). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p

    Techniques Used: Expressing, Transfection, Over Expression, Activation Assay

    Regulation of primary cilia by Intu and STAT1. a Cilium length and cell death in kidney tubules during renal IRI in mice. WT mice were subjected to sham operation or 35 min of ischemia with 48 h of reperfusion to harvest kidneys for Ac-tubulin immunofluorescence staining and TUNEL assay. TUNEL-negative tubules in injured tissues had longer cilia compared to sham controls, while TUNEL-positive tubules had shorter cilia. White triangle denotes cilia. b Ac-tubulin and LTA staining showing cilium length and frequency in proximal tubules after renal IRI in WT mice that was attenuated in PT-Intu-KO mice. c Ac-tubulin staining showing shortened cilia and lower frequency in Intu-knockdown cells (shIntu), compared to control shRNA-transfected cells (shControl). d Intu overexpression (D-REx Intu3) preserved cilium length during azide treatment. e Longer cilia and higher cilium frequency in STAT1-knockdown cells as compared to control cells. f Intu/STAT1 double knockdown cells had longer cilium than Intu only knockdown cells. g Double immunofluorescence of serine 727 phosphorylated STAT1 and γ-tubulin (centriole marker) to show that inducible Intu expression abolished centriole-associated STAT1. Fluorescence signal was quantified with ImageJ ( n = 7). n is the number of cilia ( a – f ) or centrioles ( g ). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p
    Figure Legend Snippet: Regulation of primary cilia by Intu and STAT1. a Cilium length and cell death in kidney tubules during renal IRI in mice. WT mice were subjected to sham operation or 35 min of ischemia with 48 h of reperfusion to harvest kidneys for Ac-tubulin immunofluorescence staining and TUNEL assay. TUNEL-negative tubules in injured tissues had longer cilia compared to sham controls, while TUNEL-positive tubules had shorter cilia. White triangle denotes cilia. b Ac-tubulin and LTA staining showing cilium length and frequency in proximal tubules after renal IRI in WT mice that was attenuated in PT-Intu-KO mice. c Ac-tubulin staining showing shortened cilia and lower frequency in Intu-knockdown cells (shIntu), compared to control shRNA-transfected cells (shControl). d Intu overexpression (D-REx Intu3) preserved cilium length during azide treatment. e Longer cilia and higher cilium frequency in STAT1-knockdown cells as compared to control cells. f Intu/STAT1 double knockdown cells had longer cilium than Intu only knockdown cells. g Double immunofluorescence of serine 727 phosphorylated STAT1 and γ-tubulin (centriole marker) to show that inducible Intu expression abolished centriole-associated STAT1. Fluorescence signal was quantified with ImageJ ( n = 7). n is the number of cilia ( a – f ) or centrioles ( g ). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, shRNA, Transfection, Over Expression, Marker, Expressing, Fluorescence

    STAT1 is a novel Intu-interacting protein at the basal body/centriole area. a To identify Intu-interacting proteins, Intu was transiently expressed in BUMPT cells, followed by Intu pulldown by the TAP assay. Eluted proteins from the TAP were subjected to SDS-PAGE and silver staining. One band at ~90 kDa was analyzed by mass spectrometry identifying two peptides of STAT1 (in arrowed box). b Intu constructs (or empty vector) were transiently transfected into BUMPT cells and STV-pulldown assay was performed to determine the co-precipitation of Intu and STAT1. c Intu-mycDDK was transfected into 293FT cells to collect lysate for immunoprecipitation of STAT1 with anti-STAT1 antibody or non-immune IgG (control). The precipitates were then analyzed for STAT1 and myc-tagged Intu to show their co-precipitation or interaction. d Co-immunoprecipitation of Intu and STAT1 in kidney tissues of PT-Intu-KO or WT mice after renal IRI. e Intu deletion mutants were transfected into BUMPT cells. After STV-pulldown, the Intu N-terminus containing PDZ domain (CBP-SBP-Intu ∆268-942) precipitated endogenous STAT1 while the mutants lacking this domain did not. f Intu and STAT1 interaction during IFN-γ, azide, and cisplatin treatment. 293FT cells were transfected with Intu constructs (CBP-SBP-Intu) and subjected to different treatments. Cell lysate was collected for STV-pulldown assay to show that more STAT1 was pulled down by Intu in treated 293FT cells. g Double immunofluorescence of Intu and γ-tubulin (marker of basal body/centriole) showing their co-localization in cultured mouse (BUMPT) and rat (RPTC) kidney proximal epithelial cells at different cell cycle phases. h Double immunofluorescence of Intu and Ac-tubulin (marker of cilia) showing the localization of Intu at the base of primary cilia. i Double immunofluorescence of Phospho-STAT1 S727 and γ-tubulin revealed their co-localization, while double immunofluorescence of total STAT1 and γ-tubulin showed the distribution of total STAT1 around basal body/centrioles. In three panels g – i , cells were also stained with DAPI to show nuclear morphology in merged images. Arrow-head: basal body/centriole. Scale bar: 10 µm
    Figure Legend Snippet: STAT1 is a novel Intu-interacting protein at the basal body/centriole area. a To identify Intu-interacting proteins, Intu was transiently expressed in BUMPT cells, followed by Intu pulldown by the TAP assay. Eluted proteins from the TAP were subjected to SDS-PAGE and silver staining. One band at ~90 kDa was analyzed by mass spectrometry identifying two peptides of STAT1 (in arrowed box). b Intu constructs (or empty vector) were transiently transfected into BUMPT cells and STV-pulldown assay was performed to determine the co-precipitation of Intu and STAT1. c Intu-mycDDK was transfected into 293FT cells to collect lysate for immunoprecipitation of STAT1 with anti-STAT1 antibody or non-immune IgG (control). The precipitates were then analyzed for STAT1 and myc-tagged Intu to show their co-precipitation or interaction. d Co-immunoprecipitation of Intu and STAT1 in kidney tissues of PT-Intu-KO or WT mice after renal IRI. e Intu deletion mutants were transfected into BUMPT cells. After STV-pulldown, the Intu N-terminus containing PDZ domain (CBP-SBP-Intu ∆268-942) precipitated endogenous STAT1 while the mutants lacking this domain did not. f Intu and STAT1 interaction during IFN-γ, azide, and cisplatin treatment. 293FT cells were transfected with Intu constructs (CBP-SBP-Intu) and subjected to different treatments. Cell lysate was collected for STV-pulldown assay to show that more STAT1 was pulled down by Intu in treated 293FT cells. g Double immunofluorescence of Intu and γ-tubulin (marker of basal body/centriole) showing their co-localization in cultured mouse (BUMPT) and rat (RPTC) kidney proximal epithelial cells at different cell cycle phases. h Double immunofluorescence of Intu and Ac-tubulin (marker of cilia) showing the localization of Intu at the base of primary cilia. i Double immunofluorescence of Phospho-STAT1 S727 and γ-tubulin revealed their co-localization, while double immunofluorescence of total STAT1 and γ-tubulin showed the distribution of total STAT1 around basal body/centrioles. In three panels g – i , cells were also stained with DAPI to show nuclear morphology in merged images. Arrow-head: basal body/centriole. Scale bar: 10 µm

    Techniques Used: SDS Page, Silver Staining, Mass Spectrometry, Construct, Plasmid Preparation, Transfection, Immunoprecipitation, Mouse Assay, Immunofluorescence, Marker, Cell Culture, Staining

    27) Product Images from "Recombinant Human Interferon Alpha 2b Prevents and Reverses Experimental Pulmonary Hypertension"

    Article Title: Recombinant Human Interferon Alpha 2b Prevents and Reverses Experimental Pulmonary Hypertension

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096720

    Human IFNα stimulates STAT1 phosphorylation in mice and rats. WB analysis of STAT1, phospho-STAT1 in whole lung homogenates from: (A) normoxic rats, 5 week SUH rats, and 5 week SUH rats treated with IFNα (n = 4 rats per group); or (B) normoxic mice, 6 week hypoxic mice, and 6 week hypoxic mice treated with IFNα. Densitometric ratio of phospho-STAT1 to STAT1 and phospho-STAT3 to STAT3 in lung tissue of different treatment groups in (C) SUH rats and (D) hypoxic mice.
    Figure Legend Snippet: Human IFNα stimulates STAT1 phosphorylation in mice and rats. WB analysis of STAT1, phospho-STAT1 in whole lung homogenates from: (A) normoxic rats, 5 week SUH rats, and 5 week SUH rats treated with IFNα (n = 4 rats per group); or (B) normoxic mice, 6 week hypoxic mice, and 6 week hypoxic mice treated with IFNα. Densitometric ratio of phospho-STAT1 to STAT1 and phospho-STAT3 to STAT3 in lung tissue of different treatment groups in (C) SUH rats and (D) hypoxic mice.

    Techniques Used: Mouse Assay, Western Blot

    28) Product Images from "IRF7-dependent IFN? production in response to RANKL promotes medullary thymic epithelial cell development"

    Article Title: IRF7-dependent IFN? production in response to RANKL promotes medullary thymic epithelial cell development

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1203086

    IFNβ promotes AIRE expression following RANKL stimulation a) AIRE, INS2, and CRP mRNA levels in thymic stromal cells from WT and STAT1 -/- mice (avg+/-sd; n=4) b) Thymic epithelial cells from WT mice (left) or TE-71 cells (right) were stimulated with 500 ng/mL RANKL for the indicated times and IFNβ mRNA levels were determined by qPCR (n = 4). c) Thymic epithelial cells were left untreated or pretreated with 1000 U/mL IFNβ prior to stimulation with 500 ng/mL RANKL for 24 hrs. UEA-1 + cells were analyzed for intracellular AIRE expression by flow cytometry. d) Thymic epithelial cells were depleted of CD80 +/hi cells and then stimulated with 500 ng/mL RANKL, 1000 U/mL IFNβ or both for 24hrs. Lysates were probed for AIRE, ISG15, IRF7, cFOS, p65, and Traf6 by Western blotting (representative of three experiments). e) TE-71 cells were treated as indicated, and mRNA levels for AIRE and ISG15 were determined by qPCR (avg+/-sd; n=3). f) Thymic epithelial cells were treated as indicated and mRNA levels for CRP and INS2 were determined by qPCR (avg+/-sd; n=4).
    Figure Legend Snippet: IFNβ promotes AIRE expression following RANKL stimulation a) AIRE, INS2, and CRP mRNA levels in thymic stromal cells from WT and STAT1 -/- mice (avg+/-sd; n=4) b) Thymic epithelial cells from WT mice (left) or TE-71 cells (right) were stimulated with 500 ng/mL RANKL for the indicated times and IFNβ mRNA levels were determined by qPCR (n = 4). c) Thymic epithelial cells were left untreated or pretreated with 1000 U/mL IFNβ prior to stimulation with 500 ng/mL RANKL for 24 hrs. UEA-1 + cells were analyzed for intracellular AIRE expression by flow cytometry. d) Thymic epithelial cells were depleted of CD80 +/hi cells and then stimulated with 500 ng/mL RANKL, 1000 U/mL IFNβ or both for 24hrs. Lysates were probed for AIRE, ISG15, IRF7, cFOS, p65, and Traf6 by Western blotting (representative of three experiments). e) TE-71 cells were treated as indicated, and mRNA levels for AIRE and ISG15 were determined by qPCR (avg+/-sd; n=3). f) Thymic epithelial cells were treated as indicated and mRNA levels for CRP and INS2 were determined by qPCR (avg+/-sd; n=4).

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot

    Impaired medullary thymic architecture in IFNAR -/- and STAT1 -/- mice a) Thymic architecture in WT129 and STAT1 -/- mice as revealed by H E staining of thymic sections (left panels); Keratin 5 (red) and UEA-1 (green) staining (middle panels), bar = 100μm; EpCAM (red) and AIRE (green) staining (right panels), bar = 50μm; M, medulla; C, cortex. b) Low magnification (4x) of H E stains. c) Ratio of medullary to cortical cellularity in thymic sections from WT and STAT1 -/- mice (data collected using ImageJ (NIH) and represent avg +/-SD of 3 sections each from 3 mice (p
    Figure Legend Snippet: Impaired medullary thymic architecture in IFNAR -/- and STAT1 -/- mice a) Thymic architecture in WT129 and STAT1 -/- mice as revealed by H E staining of thymic sections (left panels); Keratin 5 (red) and UEA-1 (green) staining (middle panels), bar = 100μm; EpCAM (red) and AIRE (green) staining (right panels), bar = 50μm; M, medulla; C, cortex. b) Low magnification (4x) of H E stains. c) Ratio of medullary to cortical cellularity in thymic sections from WT and STAT1 -/- mice (data collected using ImageJ (NIH) and represent avg +/-SD of 3 sections each from 3 mice (p

    Techniques Used: Mouse Assay, Staining

    29) Product Images from "Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality"

    Article Title: Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141591

    Dysregulation of regulators of proliferation and differentiation in embryonic IKK MyHC hearts. (A) The expression of Stat1, phospho-Stat1, p21 and the transgenes IKK2 and luciferase was determined by Western blot, with Erk2 shown as loading control. The diagram on the right shows a quantification of the p21 immunoblot, normalized to the expression of ERK2. (B) Expression of the indicated cell cycle modulators was determined at the mRNA level using real-time quantitative PCR in purified cardiomyocytes from IKK MyHC (black bars) and control (grey bars) embryos at E10.5. Shown are the means +SEM; N = 5, *P
    Figure Legend Snippet: Dysregulation of regulators of proliferation and differentiation in embryonic IKK MyHC hearts. (A) The expression of Stat1, phospho-Stat1, p21 and the transgenes IKK2 and luciferase was determined by Western blot, with Erk2 shown as loading control. The diagram on the right shows a quantification of the p21 immunoblot, normalized to the expression of ERK2. (B) Expression of the indicated cell cycle modulators was determined at the mRNA level using real-time quantitative PCR in purified cardiomyocytes from IKK MyHC (black bars) and control (grey bars) embryos at E10.5. Shown are the means +SEM; N = 5, *P

    Techniques Used: Expressing, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Purification

    30) Product Images from "Mechanism of HCV's resistance to IFN-? in cell culture involves expression of functional IFN-? receptor 1"

    Article Title: Mechanism of HCV's resistance to IFN-? in cell culture involves expression of functional IFN-? receptor 1

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-351

    HCV infection down-regulate IFNAR1 and induces defective Jak-Stat signaling through ER-stress mechanisms . ( A ) Cured S-5/15 Huh-7 cells were infected with cell culture derived HCV. Infected and uninfected cells were collected at different time points (0 to 96 hrs) and examined for mature form of IFNAR1 protein by Western blot analysis. ( B ) The cellular expression of IFNAR1 in the uninfected and infected Huh-7 cells was examined by Flow analysis at different time (0 to 240 hrs). The percentage of positive cells at each time point indicates the cells having the IFNAR1 expression. The peak shifts with time. ( C ) The phosphorylation of Stat1 and Stat2 protein in the HCV transfected Huh-7 cells was examined by Western blot analysis. The cells were treated with IFN-α for 30 minutes prior to lysis for detection of phosphoproteins. The beta actin in all the blots was used as loading control. ( D ) Jak-Stat signaling of HCV transfected Huh-7 cells was examined at different time points using a Firefly Luciferase activity of interferon-beta promoter. ( E ) HCV replicating Huh-7 cells were collected at different time intervals (0 to 96 hrs), protein extracts were examined for the activation of ER-stress response using panel of monoclonal and polyclonal antibodies to IRE1-α, Bip, PERK and phospho EIF2-α. Beta actin level were measured to assure, that equal amounts of proteins were loaded onto the SDS-PAGE gel are used in the Western blotting.
    Figure Legend Snippet: HCV infection down-regulate IFNAR1 and induces defective Jak-Stat signaling through ER-stress mechanisms . ( A ) Cured S-5/15 Huh-7 cells were infected with cell culture derived HCV. Infected and uninfected cells were collected at different time points (0 to 96 hrs) and examined for mature form of IFNAR1 protein by Western blot analysis. ( B ) The cellular expression of IFNAR1 in the uninfected and infected Huh-7 cells was examined by Flow analysis at different time (0 to 240 hrs). The percentage of positive cells at each time point indicates the cells having the IFNAR1 expression. The peak shifts with time. ( C ) The phosphorylation of Stat1 and Stat2 protein in the HCV transfected Huh-7 cells was examined by Western blot analysis. The cells were treated with IFN-α for 30 minutes prior to lysis for detection of phosphoproteins. The beta actin in all the blots was used as loading control. ( D ) Jak-Stat signaling of HCV transfected Huh-7 cells was examined at different time points using a Firefly Luciferase activity of interferon-beta promoter. ( E ) HCV replicating Huh-7 cells were collected at different time intervals (0 to 96 hrs), protein extracts were examined for the activation of ER-stress response using panel of monoclonal and polyclonal antibodies to IRE1-α, Bip, PERK and phospho EIF2-α. Beta actin level were measured to assure, that equal amounts of proteins were loaded onto the SDS-PAGE gel are used in the Western blotting.

    Techniques Used: Infection, Cell Culture, Derivative Assay, Western Blot, Expressing, Flow Cytometry, Transfection, Lysis, Luciferase, Activity Assay, Activation Assay, SDS Page

    Confocal microscopy showing the nuclear translocation of Stat1, Stat2 and Stat3-GFP in sensitive and resistant Huh-7 cells after IFN-α treatment . IFN-sensitive (S-5/15) and IFN-resistant (R-17/3)) cells were transfected with plasmid containing the Stat1-GFP, Stat2-GFP or Stat3-GFP. IFN-α 2 b (1000 IU/ml) was added after 48-hours of transfection and images were captured after 30 minutes of treatment. The nuclear translocation of each STAT-GFP construct with or without IFN-α treatment in the sensitive and resistant cells was examined under a confocal microscope. The images are represented as the superimposition of Green Fluorescent Protein (green), To-Pro3 633 (far red).
    Figure Legend Snippet: Confocal microscopy showing the nuclear translocation of Stat1, Stat2 and Stat3-GFP in sensitive and resistant Huh-7 cells after IFN-α treatment . IFN-sensitive (S-5/15) and IFN-resistant (R-17/3)) cells were transfected with plasmid containing the Stat1-GFP, Stat2-GFP or Stat3-GFP. IFN-α 2 b (1000 IU/ml) was added after 48-hours of transfection and images were captured after 30 minutes of treatment. The nuclear translocation of each STAT-GFP construct with or without IFN-α treatment in the sensitive and resistant cells was examined under a confocal microscope. The images are represented as the superimposition of Green Fluorescent Protein (green), To-Pro3 633 (far red).

    Techniques Used: Confocal Microscopy, Translocation Assay, Transfection, Plasmid Preparation, Construct, Microscopy

    Western blot analysis of Jak-Stat signaling between S-5/15 and R-17/3 cell line . An equal number of S-5/15 and R-17/3 Huh-7 cells were treated with IFN-α 2 b (1000 IU/ml). Cells were harvested at different time intervals (in hrs) by trypsin-EDTA treatment, washed in PBS. Western blot analysis was performed using antibodies to IFNAR1, IFNAR2, pJak1, Jak1, pTyk2, Tyk2, pStat1, Stat1, pStat2, Stat2 and β-actin and then blots were developed by ECL kit. ( A) Expression level of Jak-Stat proteins between the two cell lines. Left panel shows the expression of Jak-Stat signaling proteins in the IFN-α sensitive Huh-7 cell line (S-5/15). Right panel shows the expression of Jak-Stat signaling proteins in the IFN-α resistant Huh-7 cell line (R-17/3). The level of β-actin protein in the blot indicated that equal amounts of protein lysate were loaded onto the gel in the Western analysis. (B) Show the expression of fully glycosylated mature form of IFNAR1 (~100 kD) and IFNAR2 (~90 kD) in all nine of different IFN-α resistant cell lines and one sensitive Huh-7 cell line (S-5/15) by Western blot analysis.
    Figure Legend Snippet: Western blot analysis of Jak-Stat signaling between S-5/15 and R-17/3 cell line . An equal number of S-5/15 and R-17/3 Huh-7 cells were treated with IFN-α 2 b (1000 IU/ml). Cells were harvested at different time intervals (in hrs) by trypsin-EDTA treatment, washed in PBS. Western blot analysis was performed using antibodies to IFNAR1, IFNAR2, pJak1, Jak1, pTyk2, Tyk2, pStat1, Stat1, pStat2, Stat2 and β-actin and then blots were developed by ECL kit. ( A) Expression level of Jak-Stat proteins between the two cell lines. Left panel shows the expression of Jak-Stat signaling proteins in the IFN-α sensitive Huh-7 cell line (S-5/15). Right panel shows the expression of Jak-Stat signaling proteins in the IFN-α resistant Huh-7 cell line (R-17/3). The level of β-actin protein in the blot indicated that equal amounts of protein lysate were loaded onto the gel in the Western analysis. (B) Show the expression of fully glycosylated mature form of IFNAR1 (~100 kD) and IFNAR2 (~90 kD) in all nine of different IFN-α resistant cell lines and one sensitive Huh-7 cell line (S-5/15) by Western blot analysis.

    Techniques Used: Western Blot, Expressing

    Stable expression of IFNAR1 restored the phosphorylation Stat proteins in the cured resistant Huh-7 cell line (R-17/3) . The cured Huh-7 cell line (S-5/15) and (R-17/3) in culture were treated either with IFN-α 2 b (1000 IU/ml) or IL6 (50 ng/ml) and cells were harvested after 30 and 60 minutes. Ten micrograms of protein lysate was run on a NuPAGE 4-12% gel and immunoblotting was used to determine pStat1, pStat2 and pStat3 levels. ( A) Phosphorylation of Stat1, Stat2 and Stat3 proteins in the sensitive and resistant Huh-7 cells. Left panel shows that IFN-α induced phosphorylation Stat1, Stat2, Stat3 proteins in S-5/15 cells. Middle panel shows the lack of phosphorylation of Stat1 and Stat2 in the R-17/3 cell line after treatment with IFN-α. Right panel shows that the stable transfection of R-17/3 cells with IFNAR1 restores the Stat1 and Stat2 phosphorylation by IFN-α treatment. Total Stat1 and Stat2 levels were indistinguishable. ( B) Shows that IL-6 induced phosphorylation of Stat1 and Stat3 in Jak-Stat sensitive and defective Huh-7 cells.
    Figure Legend Snippet: Stable expression of IFNAR1 restored the phosphorylation Stat proteins in the cured resistant Huh-7 cell line (R-17/3) . The cured Huh-7 cell line (S-5/15) and (R-17/3) in culture were treated either with IFN-α 2 b (1000 IU/ml) or IL6 (50 ng/ml) and cells were harvested after 30 and 60 minutes. Ten micrograms of protein lysate was run on a NuPAGE 4-12% gel and immunoblotting was used to determine pStat1, pStat2 and pStat3 levels. ( A) Phosphorylation of Stat1, Stat2 and Stat3 proteins in the sensitive and resistant Huh-7 cells. Left panel shows that IFN-α induced phosphorylation Stat1, Stat2, Stat3 proteins in S-5/15 cells. Middle panel shows the lack of phosphorylation of Stat1 and Stat2 in the R-17/3 cell line after treatment with IFN-α. Right panel shows that the stable transfection of R-17/3 cells with IFNAR1 restores the Stat1 and Stat2 phosphorylation by IFN-α treatment. Total Stat1 and Stat2 levels were indistinguishable. ( B) Shows that IL-6 induced phosphorylation of Stat1 and Stat3 in Jak-Stat sensitive and defective Huh-7 cells.

    Techniques Used: Expressing, Stable Transfection

    31) Product Images from "Distinct Roles of Transforming Growth Factor-?-activated Kinase 1 (TAK1)-c-Rel and Interferon Regulatory Factor 4 (IRF4) Pathways in Human T Cell Lymphotropic Virus 1-transformed T helper 17 Cells Producing Interleukin-9 *"

    Article Title: Distinct Roles of Transforming Growth Factor-?-activated Kinase 1 (TAK1)-c-Rel and Interferon Regulatory Factor 4 (IRF4) Pathways in Human T Cell Lymphotropic Virus 1-transformed T helper 17 Cells Producing Interleukin-9 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.200907

    IRF4 control IFN γ by an independent pathway through IRF1. A , HuT-102 cells were transfected with IRF4 siRNA. The up-regulation of Th1-related proteins was confirmed by Western blot. B , formation of the STAT1-STAT2 heterodimer was confirmed by
    Figure Legend Snippet: IRF4 control IFN γ by an independent pathway through IRF1. A , HuT-102 cells were transfected with IRF4 siRNA. The up-regulation of Th1-related proteins was confirmed by Western blot. B , formation of the STAT1-STAT2 heterodimer was confirmed by

    Techniques Used: Transfection, Western Blot

    32) Product Images from "A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages"

    Article Title: A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages

    Journal: Genes & Development

    doi: 10.1101/gad.257592.114

    IRF8 requirement for binding of and transactivation by STAT1 in IFNβ-stimulated macrophages. ( A ) Heat map showing RNA-seq data in wild-type and Bxh2 macrophages stimulated with IFNβ as indicated. Genes were divided in three groups based
    Figure Legend Snippet: IRF8 requirement for binding of and transactivation by STAT1 in IFNβ-stimulated macrophages. ( A ) Heat map showing RNA-seq data in wild-type and Bxh2 macrophages stimulated with IFNβ as indicated. Genes were divided in three groups based

    Techniques Used: Binding Assay, RNA Sequencing Assay

    Correlation between STAT1/STAT2 and IRF8 genomic occupancy in LPS-treated macrophages. ( A ) Overlap between inducible STAT1 and STAT2 ChIP-seq peaks at 2 h after LPS stimulation. The PWMs at the right were retrieved by de novo motif discovery on either
    Figure Legend Snippet: Correlation between STAT1/STAT2 and IRF8 genomic occupancy in LPS-treated macrophages. ( A ) Overlap between inducible STAT1 and STAT2 ChIP-seq peaks at 2 h after LPS stimulation. The PWMs at the right were retrieved by de novo motif discovery on either

    Techniques Used: Chromatin Immunoprecipitation

    33) Product Images from "Kudzu Leaf Extract Suppresses the Production of Inducible Nitric Oxide Synthase, Cyclooxygenase-2, Tumor Necrosis Factor-Alpha, and Interleukin-6 via Inhibition of JNK, TBK1 and STAT1 in Inflammatory Macrophages"

    Article Title: Kudzu Leaf Extract Suppresses the Production of Inducible Nitric Oxide Synthase, Cyclooxygenase-2, Tumor Necrosis Factor-Alpha, and Interleukin-6 via Inhibition of JNK, TBK1 and STAT1 in Inflammatory Macrophages

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19051536

    Effect of kudzu leaf extract on signal transducer and activator of transcription 1 (STAT1) activation. Mouse peritoneal macrophages were treated with kudzu leaf extract for 1 h followed by stimulation with LPS (L) and IFN-γ (I) for 2 h. Whole cell extracts were prepared and subjected to Western blotting. The level of signaling proteins was analyzed using GAPDH as an internal control. One of three independent experiments is shown.
    Figure Legend Snippet: Effect of kudzu leaf extract on signal transducer and activator of transcription 1 (STAT1) activation. Mouse peritoneal macrophages were treated with kudzu leaf extract for 1 h followed by stimulation with LPS (L) and IFN-γ (I) for 2 h. Whole cell extracts were prepared and subjected to Western blotting. The level of signaling proteins was analyzed using GAPDH as an internal control. One of three independent experiments is shown.

    Techniques Used: Activation Assay, Western Blot

    Effect of robinin on LPS/IFN-γ-induced STAT1 activation in mouse peritoneal macrophages. Mouse peritoneal macrophages were treated with robinin for 1 h followed by stimulation with LPS (L) and IFN-γ (I) for 2 h. Whole cell extracts were prepared and subjected to Western blotting. The level of signaling proteins was analyzed using GAPDH as an internal control. One of three independent experiments is shown.
    Figure Legend Snippet: Effect of robinin on LPS/IFN-γ-induced STAT1 activation in mouse peritoneal macrophages. Mouse peritoneal macrophages were treated with robinin for 1 h followed by stimulation with LPS (L) and IFN-γ (I) for 2 h. Whole cell extracts were prepared and subjected to Western blotting. The level of signaling proteins was analyzed using GAPDH as an internal control. One of three independent experiments is shown.

    Techniques Used: Activation Assay, Western Blot

    34) Product Images from "The CPLANE protein Intu protects kidneys from ischemia-reperfusion injury by targeting STAT1 for degradation"

    Article Title: The CPLANE protein Intu protects kidneys from ischemia-reperfusion injury by targeting STAT1 for degradation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03628-8

    Intu targets STAT1 for degradation. a Co-transfection of Intu-mycDDK reduced the expression of FLAG-tagged STAT1α and its phosphorylation mutants (STAT1αY701F, STAT1αS727A) in 293FT cells. b Intu suppressed the expression of total and phosphorylated STAT1 during IFN-γ, azide, or cisplatin treatment of 293FT cells. c Intu or its deletion mutants were co-transfected with STAT1-FLAG into 293FT cells, showing the suppressive effect of full-length Intu and Intu ∆268-942 on STAT1 expression. d MG132 improved STAT1 expression in Intu-transfected cells. e Higher levels of total and phosphorylated STAT1, cleaved Caspase-3, and Parp, and lower levels of Bcl-2 and Bcl-xL were observed in PT-Intu-KO kidney tissues, in comparison to the wild type. Quantification was done by densitometry of immunoblots ( n = 3–4). Quantitative data are mean ± s.d. (error bars). Paired t test was used. * p
    Figure Legend Snippet: Intu targets STAT1 for degradation. a Co-transfection of Intu-mycDDK reduced the expression of FLAG-tagged STAT1α and its phosphorylation mutants (STAT1αY701F, STAT1αS727A) in 293FT cells. b Intu suppressed the expression of total and phosphorylated STAT1 during IFN-γ, azide, or cisplatin treatment of 293FT cells. c Intu or its deletion mutants were co-transfected with STAT1-FLAG into 293FT cells, showing the suppressive effect of full-length Intu and Intu ∆268-942 on STAT1 expression. d MG132 improved STAT1 expression in Intu-transfected cells. e Higher levels of total and phosphorylated STAT1, cleaved Caspase-3, and Parp, and lower levels of Bcl-2 and Bcl-xL were observed in PT-Intu-KO kidney tissues, in comparison to the wild type. Quantification was done by densitometry of immunoblots ( n = 3–4). Quantitative data are mean ± s.d. (error bars). Paired t test was used. * p

    Techniques Used: Cotransfection, Expressing, Transfection, Western Blot

    Schematic model of Intu/STAT1 regulation of cell death and cilium in kidney injury. In wild-type cells and tissues, renal IRI upregulates Intu, which binds and sequesters STAT1 in the basal body/centriole area for degradation, reducing phosphorylated STAT1 translocation to the nucleus and alleviating cell death (upper). Intu-KO cells and tissues are devoid of Intu, the STAT1 antagonizing mechanism, and as a result, more phosphorylated STAT1 accumulates in the nucleus to promote cell death (lower). Also, Intu promotes ciliogenesis, whereas STAT1 may have opposite effects. Phosphorylated STAT1 is marked with a circled P in red for Y701 and in black for S727
    Figure Legend Snippet: Schematic model of Intu/STAT1 regulation of cell death and cilium in kidney injury. In wild-type cells and tissues, renal IRI upregulates Intu, which binds and sequesters STAT1 in the basal body/centriole area for degradation, reducing phosphorylated STAT1 translocation to the nucleus and alleviating cell death (upper). Intu-KO cells and tissues are devoid of Intu, the STAT1 antagonizing mechanism, and as a result, more phosphorylated STAT1 accumulates in the nucleus to promote cell death (lower). Also, Intu promotes ciliogenesis, whereas STAT1 may have opposite effects. Phosphorylated STAT1 is marked with a circled P in red for Y701 and in black for S727

    Techniques Used: Translocation Assay

    Intu protects cells from death through STAT1. a Inducible expression of Intu suppressed STAT1 expression and Caspase-3 cleavage during azide and cisplatin treatment. b Inducible expression of Intu suppressed azide-induced cell death in STAT1α-transfected cells ( n = 5). c After exposure to cisplatin, Intu/STAT1 double overexpression cells showed resistance as compared to STAT1 single overexpression cells ( n = 8). d Confirmation of STAT1 knockdown by shRNAs in Intu/STAT1 double knockdown cells. e Knockdown of STAT1 inhibited azide-induced Caspase-3 cleavage/activation in Intu-silenced cells. f Knockdown of STAT1 rescued Intu-silenced cells from azide-induced cell death ( n = 12). g Knockdown of STAT1 rescued Intu-silenced cells from cisplatin-induced cell death ( n = 12). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p
    Figure Legend Snippet: Intu protects cells from death through STAT1. a Inducible expression of Intu suppressed STAT1 expression and Caspase-3 cleavage during azide and cisplatin treatment. b Inducible expression of Intu suppressed azide-induced cell death in STAT1α-transfected cells ( n = 5). c After exposure to cisplatin, Intu/STAT1 double overexpression cells showed resistance as compared to STAT1 single overexpression cells ( n = 8). d Confirmation of STAT1 knockdown by shRNAs in Intu/STAT1 double knockdown cells. e Knockdown of STAT1 inhibited azide-induced Caspase-3 cleavage/activation in Intu-silenced cells. f Knockdown of STAT1 rescued Intu-silenced cells from azide-induced cell death ( n = 12). g Knockdown of STAT1 rescued Intu-silenced cells from cisplatin-induced cell death ( n = 12). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p

    Techniques Used: Expressing, Transfection, Over Expression, Activation Assay

    Regulation of primary cilia by Intu and STAT1. a Cilium length and cell death in kidney tubules during renal IRI in mice. WT mice were subjected to sham operation or 35 min of ischemia with 48 h of reperfusion to harvest kidneys for Ac-tubulin immunofluorescence staining and TUNEL assay. TUNEL-negative tubules in injured tissues had longer cilia compared to sham controls, while TUNEL-positive tubules had shorter cilia. White triangle denotes cilia. b Ac-tubulin and LTA staining showing cilium length and frequency in proximal tubules after renal IRI in WT mice that was attenuated in PT-Intu-KO mice. c Ac-tubulin staining showing shortened cilia and lower frequency in Intu-knockdown cells (shIntu), compared to control shRNA-transfected cells (shControl). d Intu overexpression (D-REx Intu3) preserved cilium length during azide treatment. e Longer cilia and higher cilium frequency in STAT1-knockdown cells as compared to control cells. f Intu/STAT1 double knockdown cells had longer cilium than Intu only knockdown cells. g Double immunofluorescence of serine 727 phosphorylated STAT1 and γ-tubulin (centriole marker) to show that inducible Intu expression abolished centriole-associated STAT1. Fluorescence signal was quantified with ImageJ ( n = 7). n is the number of cilia ( a – f ) or centrioles ( g ). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p
    Figure Legend Snippet: Regulation of primary cilia by Intu and STAT1. a Cilium length and cell death in kidney tubules during renal IRI in mice. WT mice were subjected to sham operation or 35 min of ischemia with 48 h of reperfusion to harvest kidneys for Ac-tubulin immunofluorescence staining and TUNEL assay. TUNEL-negative tubules in injured tissues had longer cilia compared to sham controls, while TUNEL-positive tubules had shorter cilia. White triangle denotes cilia. b Ac-tubulin and LTA staining showing cilium length and frequency in proximal tubules after renal IRI in WT mice that was attenuated in PT-Intu-KO mice. c Ac-tubulin staining showing shortened cilia and lower frequency in Intu-knockdown cells (shIntu), compared to control shRNA-transfected cells (shControl). d Intu overexpression (D-REx Intu3) preserved cilium length during azide treatment. e Longer cilia and higher cilium frequency in STAT1-knockdown cells as compared to control cells. f Intu/STAT1 double knockdown cells had longer cilium than Intu only knockdown cells. g Double immunofluorescence of serine 727 phosphorylated STAT1 and γ-tubulin (centriole marker) to show that inducible Intu expression abolished centriole-associated STAT1. Fluorescence signal was quantified with ImageJ ( n = 7). n is the number of cilia ( a – f ) or centrioles ( g ). Quantitative data are mean ± s.d. (error bars). Grouped t test was used. * p

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, TUNEL Assay, shRNA, Transfection, Over Expression, Marker, Expressing, Fluorescence

    STAT1 is a novel Intu-interacting protein at the basal body/centriole area. a To identify Intu-interacting proteins, Intu was transiently expressed in BUMPT cells, followed by Intu pulldown by the TAP assay. Eluted proteins from the TAP were subjected to SDS-PAGE and silver staining. One band at ~90 kDa was analyzed by mass spectrometry identifying two peptides of STAT1 (in arrowed box). b Intu constructs (or empty vector) were transiently transfected into BUMPT cells and STV-pulldown assay was performed to determine the co-precipitation of Intu and STAT1. c Intu-mycDDK was transfected into 293FT cells to collect lysate for immunoprecipitation of STAT1 with anti-STAT1 antibody or non-immune IgG (control). The precipitates were then analyzed for STAT1 and myc-tagged Intu to show their co-precipitation or interaction. d Co-immunoprecipitation of Intu and STAT1 in kidney tissues of PT-Intu-KO or WT mice after renal IRI. e Intu deletion mutants were transfected into BUMPT cells. After STV-pulldown, the Intu N-terminus containing PDZ domain (CBP-SBP-Intu ∆268-942) precipitated endogenous STAT1 while the mutants lacking this domain did not. f Intu and STAT1 interaction during IFN-γ, azide, and cisplatin treatment. 293FT cells were transfected with Intu constructs (CBP-SBP-Intu) and subjected to different treatments. Cell lysate was collected for STV-pulldown assay to show that more STAT1 was pulled down by Intu in treated 293FT cells. g Double immunofluorescence of Intu and γ-tubulin (marker of basal body/centriole) showing their co-localization in cultured mouse (BUMPT) and rat (RPTC) kidney proximal epithelial cells at different cell cycle phases. h Double immunofluorescence of Intu and Ac-tubulin (marker of cilia) showing the localization of Intu at the base of primary cilia. i Double immunofluorescence of Phospho-STAT1 S727 and γ-tubulin revealed their co-localization, while double immunofluorescence of total STAT1 and γ-tubulin showed the distribution of total STAT1 around basal body/centrioles. In three panels g – i , cells were also stained with DAPI to show nuclear morphology in merged images. Arrow-head: basal body/centriole. Scale bar: 10 µm
    Figure Legend Snippet: STAT1 is a novel Intu-interacting protein at the basal body/centriole area. a To identify Intu-interacting proteins, Intu was transiently expressed in BUMPT cells, followed by Intu pulldown by the TAP assay. Eluted proteins from the TAP were subjected to SDS-PAGE and silver staining. One band at ~90 kDa was analyzed by mass spectrometry identifying two peptides of STAT1 (in arrowed box). b Intu constructs (or empty vector) were transiently transfected into BUMPT cells and STV-pulldown assay was performed to determine the co-precipitation of Intu and STAT1. c Intu-mycDDK was transfected into 293FT cells to collect lysate for immunoprecipitation of STAT1 with anti-STAT1 antibody or non-immune IgG (control). The precipitates were then analyzed for STAT1 and myc-tagged Intu to show their co-precipitation or interaction. d Co-immunoprecipitation of Intu and STAT1 in kidney tissues of PT-Intu-KO or WT mice after renal IRI. e Intu deletion mutants were transfected into BUMPT cells. After STV-pulldown, the Intu N-terminus containing PDZ domain (CBP-SBP-Intu ∆268-942) precipitated endogenous STAT1 while the mutants lacking this domain did not. f Intu and STAT1 interaction during IFN-γ, azide, and cisplatin treatment. 293FT cells were transfected with Intu constructs (CBP-SBP-Intu) and subjected to different treatments. Cell lysate was collected for STV-pulldown assay to show that more STAT1 was pulled down by Intu in treated 293FT cells. g Double immunofluorescence of Intu and γ-tubulin (marker of basal body/centriole) showing their co-localization in cultured mouse (BUMPT) and rat (RPTC) kidney proximal epithelial cells at different cell cycle phases. h Double immunofluorescence of Intu and Ac-tubulin (marker of cilia) showing the localization of Intu at the base of primary cilia. i Double immunofluorescence of Phospho-STAT1 S727 and γ-tubulin revealed their co-localization, while double immunofluorescence of total STAT1 and γ-tubulin showed the distribution of total STAT1 around basal body/centrioles. In three panels g – i , cells were also stained with DAPI to show nuclear morphology in merged images. Arrow-head: basal body/centriole. Scale bar: 10 µm

    Techniques Used: SDS Page, Silver Staining, Mass Spectrometry, Construct, Plasmid Preparation, Transfection, Immunoprecipitation, Mouse Assay, Immunofluorescence, Marker, Cell Culture, Staining

    35) Product Images from "CD40 Induces Antigen Transporter and Immunoproteasome Gene Expression in Carcinomas via the Coordinated Action of NF-?B and of NF-?B-Mediated De Novo Synthesis of IRF-1 ▿"

    Article Title: CD40 Induces Antigen Transporter and Immunoproteasome Gene Expression in Carcinomas via the Coordinated Action of NF-?B and of NF-?B-Mediated De Novo Synthesis of IRF-1 ▿

    Journal:

    doi: 10.1128/MCB.00611-08

    CD40 ligation induces MAPK and NF-κB but not STAT1 signaling prior to the induction of IRF-1 expression. (A) EJ cells were stimulated with 0.5 μg of CD40L/ml for 7 or 15 min or left untreated, and cell lysates were analyzed for the expression
    Figure Legend Snippet: CD40 ligation induces MAPK and NF-κB but not STAT1 signaling prior to the induction of IRF-1 expression. (A) EJ cells were stimulated with 0.5 μg of CD40L/ml for 7 or 15 min or left untreated, and cell lysates were analyzed for the expression

    Techniques Used: Ligation, Expressing

    36) Product Images from "Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells"

    Article Title: Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

    Journal: Blood

    doi: 10.1182/blood-2007-03-081448

    G-CSF induced STAT3 signaling and Gfi-1 expression. (A) Western blot analysis of STAT1, STAT3, and STAT5 phosphorylation in cell lysates of 32Dcl3 cells cultured for 1 to 144 hours with IL-3 alone or with IL-3 plus G-CSF. Blots were first stained for
    Figure Legend Snippet: G-CSF induced STAT3 signaling and Gfi-1 expression. (A) Western blot analysis of STAT1, STAT3, and STAT5 phosphorylation in cell lysates of 32Dcl3 cells cultured for 1 to 144 hours with IL-3 alone or with IL-3 plus G-CSF. Blots were first stained for

    Techniques Used: Expressing, Western Blot, Cell Culture, Staining

    37) Product Images from "SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo"

    Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.788877

    IFN-α– but not IFN-λ–induced STAT1 phosphorylation becomes refractory to continuous stimulation. A , liver biopsies from chronic hepatitis C patients ( n = 16) were divided into three groups based on their IFNL4 genotype (rs368234815; TT/TT, TT/dG, and dG/dG). Total RNA from biopsies and Huh7 and Huh7 LR cells were prepared. Expression of the IFNLR1 transcript was analyzed by quantitative PCR. Results (mean ± S.D.) are shown as copy numbers per 40 ng of total RNA. B and C , Huh7 LR cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for the indicated times. B , p-STAT1, STAT1, p-STAT2, STAT2, p-STAT3, STAT3, USP18, SOCS1, SOCS3, and actin were visualized using specific antibodies. Shown are representative blots from two independent experiments. C , transcripts of interferon-stimulated genes ( RSAD2 , IFI27 , and GBP5 ) were quantified by PCR. Results (mean ± S.D., n = 3) are shown as relative expression to GAPDH. ut , untreated.
    Figure Legend Snippet: IFN-α– but not IFN-λ–induced STAT1 phosphorylation becomes refractory to continuous stimulation. A , liver biopsies from chronic hepatitis C patients ( n = 16) were divided into three groups based on their IFNL4 genotype (rs368234815; TT/TT, TT/dG, and dG/dG). Total RNA from biopsies and Huh7 and Huh7 LR cells were prepared. Expression of the IFNLR1 transcript was analyzed by quantitative PCR. Results (mean ± S.D.) are shown as copy numbers per 40 ng of total RNA. B and C , Huh7 LR cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for the indicated times. B , p-STAT1, STAT1, p-STAT2, STAT2, p-STAT3, STAT3, USP18, SOCS1, SOCS3, and actin were visualized using specific antibodies. Shown are representative blots from two independent experiments. C , transcripts of interferon-stimulated genes ( RSAD2 , IFI27 , and GBP5 ) were quantified by PCR. Results (mean ± S.D., n = 3) are shown as relative expression to GAPDH. ut , untreated.

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

    Depletion of Socs-1 increased IFN-λ–induced ISGs expression in vivo . Control and Socs1 −/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2 , Oas1 , Stat1 , Usp18 , and Socs1 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19 . Three to four animals were used per time point and condition. Unpaired t test with Welch's correction; *, p
    Figure Legend Snippet: Depletion of Socs-1 increased IFN-λ–induced ISGs expression in vivo . Control and Socs1 −/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2 , Oas1 , Stat1 , Usp18 , and Socs1 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19 . Three to four animals were used per time point and condition. Unpaired t test with Welch's correction; *, p

    Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Real-time Polymerase Chain Reaction

    Overexpression of SOCS1, SOCS3, and USP18 leads to a reduction of IFN-α– and IFN-λ–mediated STAT1 phosphorylation. A , Huh7 LR cells were transiently transfected with control, SOCS1, SOCS3, or USP18 expression plasmids. 24 h later, cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for 30 min, and p-STAT1, STAT1, SOCS1, SOCS3, USP18, and actin were visualized by immunoblotting. Shown are representative blots from three independent experiments. B , Huh7 LR cells were transfected with SOCS1, SOCS3, or USP18 expression plasmids for 24 h. The mRNA expression levels of SOCS1 , SOCS3 , and USP18 were analyzed by quantitative PCR and compared with the endogenously induced SOCS1 , SOCS3 , or USP18 upon IFN-α or IFN-λ1 stimulation at the indicated time points. The results (mean ± S.D., n = 3) are shown as relative expression to GAPDH . Protein levels of SOCS1, SOCS3, and USP18 and actin were visualized using specific antibodies. ox , overexpression; ut , untreated.
    Figure Legend Snippet: Overexpression of SOCS1, SOCS3, and USP18 leads to a reduction of IFN-α– and IFN-λ–mediated STAT1 phosphorylation. A , Huh7 LR cells were transiently transfected with control, SOCS1, SOCS3, or USP18 expression plasmids. 24 h later, cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for 30 min, and p-STAT1, STAT1, SOCS1, SOCS3, USP18, and actin were visualized by immunoblotting. Shown are representative blots from three independent experiments. B , Huh7 LR cells were transfected with SOCS1, SOCS3, or USP18 expression plasmids for 24 h. The mRNA expression levels of SOCS1 , SOCS3 , and USP18 were analyzed by quantitative PCR and compared with the endogenously induced SOCS1 , SOCS3 , or USP18 upon IFN-α or IFN-λ1 stimulation at the indicated time points. The results (mean ± S.D., n = 3) are shown as relative expression to GAPDH . Protein levels of SOCS1, SOCS3, and USP18 and actin were visualized using specific antibodies. ox , overexpression; ut , untreated.

    Techniques Used: Over Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction

    Depletion of Usp18 increased IFN-α–induced ISGs expression in vivo . Control and Usp18 −/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2 , Oas1 , Stat1 , Socs1 , and Usp18 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19 . Three to five animals were used per time point and condition. Unpaired t test with Welch's correction; *, p
    Figure Legend Snippet: Depletion of Usp18 increased IFN-α–induced ISGs expression in vivo . Control and Usp18 −/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2 , Oas1 , Stat1 , Socs1 , and Usp18 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19 . Three to five animals were used per time point and condition. Unpaired t test with Welch's correction; *, p

    Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Real-time Polymerase Chain Reaction

    38) Product Images from "Roles of phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase in the regulation of protein kinase C-? activation in interferon-?-stimulated macrophages"

    Article Title: Roles of phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase in the regulation of protein kinase C-? activation in interferon-?-stimulated macrophages

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2009.03055.x

    Interferon-γ (IFN-γ) induces the association of protein kinase C-α (PKC-α) with signal transducer and activator of transcription 1 (STAT1). Adherent bone marrow-derived macrophages (BMDM) were incubated in the absence or presence of 100 U/ml of IFN-γ for the indicated periods of time. PKC-α was immunoprecipitated and the presence of STAT1 in immunoprecipitates was determined by Western blot analysis, as described in the Materials and methods . The blot was stripped and reprobed with an anti-PKC-α immunoglobulin (IgG2b) to ensure that equal amounts of PKC-α were immunoprecipitated in each sample. Similar results were obtained in two independent experiments.
    Figure Legend Snippet: Interferon-γ (IFN-γ) induces the association of protein kinase C-α (PKC-α) with signal transducer and activator of transcription 1 (STAT1). Adherent bone marrow-derived macrophages (BMDM) were incubated in the absence or presence of 100 U/ml of IFN-γ for the indicated periods of time. PKC-α was immunoprecipitated and the presence of STAT1 in immunoprecipitates was determined by Western blot analysis, as described in the Materials and methods . The blot was stripped and reprobed with an anti-PKC-α immunoglobulin (IgG2b) to ensure that equal amounts of PKC-α were immunoprecipitated in each sample. Similar results were obtained in two independent experiments.

    Techniques Used: Derivative Assay, Incubation, Immunoprecipitation, Western Blot

    39) Product Images from "Depletion of Cellular Glutathione Modulates LIF-Induced JAK1-STAT3 Signaling in Cardiac Myocytes"

    Article Title: Depletion of Cellular Glutathione Modulates LIF-Induced JAK1-STAT3 Signaling in Cardiac Myocytes

    Journal: The international journal of biochemistry & cell biology

    doi: 10.1016/j.biocel.2012.08.016

    Differential response of STAT1 and STAT3 to crosslinking agents. (A B) Aliquots of mouse heart homogenates were incubated with vehicle, 500 μM NCP, 1 mM diamide, or 500 μM NCP + 1 mM diamide for 30 min. Samples were processed for SDS-PAGE and Western analysis in nonreducing or reducing sample buffer. (A) Membranes were probed for STAT3 and STAT1 using the Li-COR Odyssey detection system. (B) For STAT1 and STAT3, the intensity of the band in the nonreduced sample was normalized to the intensity of the band after reduction and expressed relative to the total oxidized sample (NCP + diamide). ***P
    Figure Legend Snippet: Differential response of STAT1 and STAT3 to crosslinking agents. (A B) Aliquots of mouse heart homogenates were incubated with vehicle, 500 μM NCP, 1 mM diamide, or 500 μM NCP + 1 mM diamide for 30 min. Samples were processed for SDS-PAGE and Western analysis in nonreducing or reducing sample buffer. (A) Membranes were probed for STAT3 and STAT1 using the Li-COR Odyssey detection system. (B) For STAT1 and STAT3, the intensity of the band in the nonreduced sample was normalized to the intensity of the band after reduction and expressed relative to the total oxidized sample (NCP + diamide). ***P

    Techniques Used: Incubation, SDS Page, Western Blot

    GSH depletion attenuates LIF-induced ERK1/2, but not STAT1 activation. (A) Cardiac myocytes that were treated with various concentrations of BSO were dosed with 2 ng/mL LIF for 10 min. Cell extracts were prepared and analyzed for activated ERK1/2 (pERK1/2) and total ERK1/2 as a loading control. Relative levels of inhibition were determined. *P
    Figure Legend Snippet: GSH depletion attenuates LIF-induced ERK1/2, but not STAT1 activation. (A) Cardiac myocytes that were treated with various concentrations of BSO were dosed with 2 ng/mL LIF for 10 min. Cell extracts were prepared and analyzed for activated ERK1/2 (pERK1/2) and total ERK1/2 as a loading control. Relative levels of inhibition were determined. *P

    Techniques Used: Activation Assay, Inhibition

    40) Product Images from "Growth Hormone Did Not Activate Its Intracellular Signaling Molecules in Rats’ Liver Hepatocytes During Early Life Period"

    Article Title: Growth Hormone Did Not Activate Its Intracellular Signaling Molecules in Rats’ Liver Hepatocytes During Early Life Period

    Journal: International Journal of Endocrinology and Metabolism

    doi: 10.5812/ijem.61385

    Abundance of phosphorylated proteins in livers of different age group rats (1-day-old, 4-day-old, 1-week-old, 2-week-old and 3-week-old) in response to GH A, phosphorylated STAT5 protein amount; B, phosphorylated STAT3 protein amount; C, phosphorylated STAT1 protein amount. The rats were injected with GH (2 mg/kg of body weight) (filled bars) or saline (open bars) in the inferior vena cava. To determine protein activation, equal amounts of solubilized liver protein were assessed by Western blots. Results are expressed as mean ± SD of three separate experiments. *p
    Figure Legend Snippet: Abundance of phosphorylated proteins in livers of different age group rats (1-day-old, 4-day-old, 1-week-old, 2-week-old and 3-week-old) in response to GH A, phosphorylated STAT5 protein amount; B, phosphorylated STAT3 protein amount; C, phosphorylated STAT1 protein amount. The rats were injected with GH (2 mg/kg of body weight) (filled bars) or saline (open bars) in the inferior vena cava. To determine protein activation, equal amounts of solubilized liver protein were assessed by Western blots. Results are expressed as mean ± SD of three separate experiments. *p

    Techniques Used: Injection, Activation Assay, Western Blot

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    Article Snippet: .. The cell lysates were subjected to immunoprecipitation and immunoblot as previously described ( ) using antibodies of phospho-STAT1 (pSTAT1) (Tyr-701), pSTAT3 (Tyr-705), STAT1, and STAT3 (Cell Signaling Technology, Inc., Danvers, MA), MKP-1 and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and iNOS, (BD Biosciences, San Jose, CA). .. For quantitation of the changes in STAT1 phosphorylation, the intensities of the bands representing STAT1 and pSTAT1 (Tyr-701) were measured by densitometry using an image scanner (EPSON GT-8000) and National Institutes of Health Image software.

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    Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization
    Article Snippet: The primary antibodies against p-JNK (Thr183/Tyr185), JNK, p-p38 (Thr180/Tyr182), p38, p-STAT1 (Ser727), STAT1, p-ERK (Thr202/Tyr204), ERK, p-p65 (Ser536), p65 and H3 were purchased from Cell Signaling Technology.

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    Cell Signaling Technology Inc rabbit polyclonal antibodies against tyr 701 phosphorylated stat1
    <t>U-STAT1,</t> U-STAT2, and IRF9 form U-ISGF3, which binds to ISREs on the target gene promoters. hTERT-HME1 cells expressing high levels of U-STAT1, U-STAT2, and IRF9 without IFN stimulation were analysed by co-immunoprecipitation (Co-IP) and chromatin-immunoprecipitation (ChIP) assays. ( A ) Nuclear proteins were used for Co-IP with normal rabbit IgG or rabbit <t>polyclonal</t> antibodies against STAT1, STAT2, or IRF9. To stabilize protein–protein interactions, the nuclear fraction was treated with the cleavable cross-linker, dimethyl-3,3′-dithiobis-propinimidate (DTBP, lanes 1, 2, and 4). Mouse monoclonal antibodies against STAT1, STAT2, or IRF9 were used for the western method. The purity of the fractions was assessed by determining the levels of GAPDH (a cytoplasmic protein) and HDAC1 (a nuclear protein) in the input lysates by the western method. ( B , C ) Total protein lysates were cross-linked with 1% formaldehyde and the cell lysates were cross-linked with DTBP. Chromatin was sheared into
    Rabbit Polyclonal Antibodies Against Tyr 701 Phosphorylated Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit monoclonal phospho stat1
    Esophageal cells with mutant p53 R175H and POSTN reveal activation of the <t>STAT1</t> signaling pathway. ( a ) Venn diagram displaying the number of genes with significant differential expression between the compared groups. Gene expression data were generated with RNA isolated from dissected epithelia of EPC-hTERT-p53 R175H -POSTN cells grown in organotypic culture ( n =3) compared with EPC-hTERT-p53 R175H -neo cells ( n =3) as well as parental non-invading EPC-hTERT cells ( n =3). The blue circle (gene lists hTERT and p53 R175H ) represents genes differentially expressed between EPC-hTERT and EPC-hTERT-p53 R175H -neo (3121). The red circle (gene lists p53 R175H and POSTN) represents genes differentially expressed between EPC-hTERT-p53 R175H -neo and EPC-hTERT-p53 R175H -POSTN (1808). ( P
    Rabbit Monoclonal Phospho Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    promega total stat1
    5′ppp-RNA activation of RIG-I stimulates the <t>IRF3–IFNβ–IFNAR–STAT1–IFIT1</t> pathway to induce cell death of A549 cells. Cells were transfected with Lyovec control or 1 μg/ml of 5′ppp-RNA with Lyovec. b , d , f Lysates were collected at indicated times after 5′ppp-RNA transfection and assayed for the indicated proteins. Blots are representative of three independent experiments. a , c , e , g Cell viability was determined using Cell Titer assay and normalized to the Lyovec control after 72 h. Specifically, cells were a treated with human recombinant IFNβ for the stated times, b , c pre-and cotreated with 100 or 200 μM Amlexanox, a TBK1 antagonist 1 h prior to 5′ppp-RNA transfection, d , e pre-and co-treated with 0.1 μg/ml B18R an IFNAR antagonist 1 h prior to 5′ppp-RNA transfection, and f , g transfected with negative siCTRL and siIFIT1 16 h post-transfection of 5′ppp-RNA. Data is represented as mean ± SEM of n = 3 independent experiments. * P
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    Cell Signaling Technology Inc non phosphorylated stat1
    Effect of IL-26 on <t>STAT1,</t> STAT3, and STAT6 activation in M1 and M2 macrophage differentiation. ( A ) RAW264.7 cells were serum-starved for 16 h and treated with IFN-γ (20 ng/mL) or IL-4 (20 ng/mL) in the presence or absence of IL-26, and ( B ) THP-1 cells were treated with IFN-γ (20 ng/mL) plus LPS (10 ng/mL) or IL-4 (20 ng/mL) plus IL-13 (20 ng/mL) in the presence or absence of IL-26 for 2 and 4 h to further detect phosphorylated or non-phosphorylated STAT1, STAT3, and STAT6 proteins. Cell extracts were analyzed by western blot using antibodies specifically directed against the phosphorylated forms of STATs, compared with data obtained with antibodies directed against the unphosphorylated states of the kinases. Equal amounts of protein were loaded in each lane as demonstrated by the level of GAPDH. A representative result of at least three independent experiments is shown. (FL+IL-26: IFN-γ+LPS+IL-26; 413+IL-26: IL-4 + IL-13+IL-26)
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    U-STAT1, U-STAT2, and IRF9 form U-ISGF3, which binds to ISREs on the target gene promoters. hTERT-HME1 cells expressing high levels of U-STAT1, U-STAT2, and IRF9 without IFN stimulation were analysed by co-immunoprecipitation (Co-IP) and chromatin-immunoprecipitation (ChIP) assays. ( A ) Nuclear proteins were used for Co-IP with normal rabbit IgG or rabbit polyclonal antibodies against STAT1, STAT2, or IRF9. To stabilize protein–protein interactions, the nuclear fraction was treated with the cleavable cross-linker, dimethyl-3,3′-dithiobis-propinimidate (DTBP, lanes 1, 2, and 4). Mouse monoclonal antibodies against STAT1, STAT2, or IRF9 were used for the western method. The purity of the fractions was assessed by determining the levels of GAPDH (a cytoplasmic protein) and HDAC1 (a nuclear protein) in the input lysates by the western method. ( B , C ) Total protein lysates were cross-linked with 1% formaldehyde and the cell lysates were cross-linked with DTBP. Chromatin was sheared into

    Journal: The EMBO Journal

    Article Title: IFN?-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage

    doi: 10.1038/emboj.2013.203

    Figure Lengend Snippet: U-STAT1, U-STAT2, and IRF9 form U-ISGF3, which binds to ISREs on the target gene promoters. hTERT-HME1 cells expressing high levels of U-STAT1, U-STAT2, and IRF9 without IFN stimulation were analysed by co-immunoprecipitation (Co-IP) and chromatin-immunoprecipitation (ChIP) assays. ( A ) Nuclear proteins were used for Co-IP with normal rabbit IgG or rabbit polyclonal antibodies against STAT1, STAT2, or IRF9. To stabilize protein–protein interactions, the nuclear fraction was treated with the cleavable cross-linker, dimethyl-3,3′-dithiobis-propinimidate (DTBP, lanes 1, 2, and 4). Mouse monoclonal antibodies against STAT1, STAT2, or IRF9 were used for the western method. The purity of the fractions was assessed by determining the levels of GAPDH (a cytoplasmic protein) and HDAC1 (a nuclear protein) in the input lysates by the western method. ( B , C ) Total protein lysates were cross-linked with 1% formaldehyde and the cell lysates were cross-linked with DTBP. Chromatin was sheared into

    Article Snippet: Mouse monoclonal antibodies against STAT1 (BD Transduction), STAT2, IRF9 (ISGF3γ), and IRF1 (Santa Cruz Biotechnology), and rabbit polyclonal antibodies against Tyr 701-phosphorylated STAT1, Tyr 690-phophorylated STAT2 (Cell Signaling), and STAT1 (Upstate) were used for western analyses.

    Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Western Blot

    Esophageal cells with mutant p53 R175H and POSTN reveal activation of the STAT1 signaling pathway. ( a ) Venn diagram displaying the number of genes with significant differential expression between the compared groups. Gene expression data were generated with RNA isolated from dissected epithelia of EPC-hTERT-p53 R175H -POSTN cells grown in organotypic culture ( n =3) compared with EPC-hTERT-p53 R175H -neo cells ( n =3) as well as parental non-invading EPC-hTERT cells ( n =3). The blue circle (gene lists hTERT and p53 R175H ) represents genes differentially expressed between EPC-hTERT and EPC-hTERT-p53 R175H -neo (3121). The red circle (gene lists p53 R175H and POSTN) represents genes differentially expressed between EPC-hTERT-p53 R175H -neo and EPC-hTERT-p53 R175H -POSTN (1808). ( P

    Journal: Oncogenesis

    Article Title: Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment

    doi: 10.1038/oncsis.2013.17

    Figure Lengend Snippet: Esophageal cells with mutant p53 R175H and POSTN reveal activation of the STAT1 signaling pathway. ( a ) Venn diagram displaying the number of genes with significant differential expression between the compared groups. Gene expression data were generated with RNA isolated from dissected epithelia of EPC-hTERT-p53 R175H -POSTN cells grown in organotypic culture ( n =3) compared with EPC-hTERT-p53 R175H -neo cells ( n =3) as well as parental non-invading EPC-hTERT cells ( n =3). The blue circle (gene lists hTERT and p53 R175H ) represents genes differentially expressed between EPC-hTERT and EPC-hTERT-p53 R175H -neo (3121). The red circle (gene lists p53 R175H and POSTN) represents genes differentially expressed between EPC-hTERT-p53 R175H -neo and EPC-hTERT-p53 R175H -POSTN (1808). ( P

    Article Snippet: For immunohistochemistry, rabbit polyclonal POSTN (Abcam, ab 14041) and rabbit monoclonal phospho-STAT1 (Tyr701; Cell Signaling) were used.

    Techniques: Mutagenesis, Activation Assay, Expressing, Generated, Isolation

    STAT1 knockdown in EPC-hTERT-p53 R175H -POSTN and transformed EPC-hTERT-EGFR-p53 R175H cells show decrease in invasion. ( a ) Western blot confirming knockdown total STAT1 and STAT1 phosphorylation in invasive EPC-hTERT-p53 R175H -POSTN and in transformed, genetically engineered EPC-hTERT-EGFR-p53 R175H cells using two independent shRNAs directed against STAT1 and non-specific shRNAs as controls (A and B represent independently generated cell lines with the same genotype). GAPDH was used as a loading control. ( b ) Transwell Boyden Chamber invasion assay of EPC-hTERT-p53 R175H -POSTN-shSTAT1-A and -B and EPC-hTERT-EGFR-p53 R175H -shSTAT1-A and -B cells compared with control EPC-hTERT-p53 R175H -POSTN-shNS-A and -B and EPC-hTERT-EGFR-p53 R175H -shNS-A and -B cells. Bar graphs represent fold changes±s.e.m. * P

    Journal: Oncogenesis

    Article Title: Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment

    doi: 10.1038/oncsis.2013.17

    Figure Lengend Snippet: STAT1 knockdown in EPC-hTERT-p53 R175H -POSTN and transformed EPC-hTERT-EGFR-p53 R175H cells show decrease in invasion. ( a ) Western blot confirming knockdown total STAT1 and STAT1 phosphorylation in invasive EPC-hTERT-p53 R175H -POSTN and in transformed, genetically engineered EPC-hTERT-EGFR-p53 R175H cells using two independent shRNAs directed against STAT1 and non-specific shRNAs as controls (A and B represent independently generated cell lines with the same genotype). GAPDH was used as a loading control. ( b ) Transwell Boyden Chamber invasion assay of EPC-hTERT-p53 R175H -POSTN-shSTAT1-A and -B and EPC-hTERT-EGFR-p53 R175H -shSTAT1-A and -B cells compared with control EPC-hTERT-p53 R175H -POSTN-shNS-A and -B and EPC-hTERT-EGFR-p53 R175H -shNS-A and -B cells. Bar graphs represent fold changes±s.e.m. * P

    Article Snippet: For immunohistochemistry, rabbit polyclonal POSTN (Abcam, ab 14041) and rabbit monoclonal phospho-STAT1 (Tyr701; Cell Signaling) were used.

    Techniques: Transformation Assay, Western Blot, Generated, Invasion Assay

    Inducible knockdown of POSTN in ESCC xenograft tumors display decreased p53 expression and STAT1 activation. ( a ) Phospho-STAT1(Tyr701) expression by immunohistochemistry of tumors formed in vivo by subcutaneous injection of HCE4 cancer cells stably transfected with either lentiviral doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) vectors. Left panels represent tumors that were not induced with doxycycline (DOX), and right panels represent tumors induced with doxycycline. Bar=100 μℳ. ( b ) Phospho-STAT1(Tyr701) expression by immunohistochemistry of tumors formed in vivo by subcutaneous injection of TE-11 cancer cells stably transfected with either lentiviral doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) vectors. Left panels represent tumors that were not induced with doxycycline, and right panels represent tumors induced with doxycycline. Bar=100 μℳ. ( c ) Western blot analysis of STAT1 and p53 expression in four pairs of lysates isolated from HCE4 xenograft tumors transduced with doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) with or without doxycycline treatment. Immunoblotting for POSTN expression to confirm doxycycline induced knockdown. GAPDH was used as a loading control. ( d ) Western blot analysis of STAT1 and p53 expression in four pairs of lysates isolated from TE-11 xenograft tumors transduced with doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) with or without doxycycline treatment. Immunoblotting for POSTN expression to confirm doxycycline induced knockdown. GAPDH was used as a loading control.

    Journal: Oncogenesis

    Article Title: Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment

    doi: 10.1038/oncsis.2013.17

    Figure Lengend Snippet: Inducible knockdown of POSTN in ESCC xenograft tumors display decreased p53 expression and STAT1 activation. ( a ) Phospho-STAT1(Tyr701) expression by immunohistochemistry of tumors formed in vivo by subcutaneous injection of HCE4 cancer cells stably transfected with either lentiviral doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) vectors. Left panels represent tumors that were not induced with doxycycline (DOX), and right panels represent tumors induced with doxycycline. Bar=100 μℳ. ( b ) Phospho-STAT1(Tyr701) expression by immunohistochemistry of tumors formed in vivo by subcutaneous injection of TE-11 cancer cells stably transfected with either lentiviral doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) vectors. Left panels represent tumors that were not induced with doxycycline, and right panels represent tumors induced with doxycycline. Bar=100 μℳ. ( c ) Western blot analysis of STAT1 and p53 expression in four pairs of lysates isolated from HCE4 xenograft tumors transduced with doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) with or without doxycycline treatment. Immunoblotting for POSTN expression to confirm doxycycline induced knockdown. GAPDH was used as a loading control. ( d ) Western blot analysis of STAT1 and p53 expression in four pairs of lysates isolated from TE-11 xenograft tumors transduced with doxycycline-inducible non-specific targeting shRNA (shNS) or shRNA specific to periostin (shPOSTN) with or without doxycycline treatment. Immunoblotting for POSTN expression to confirm doxycycline induced knockdown. GAPDH was used as a loading control.

    Article Snippet: For immunohistochemistry, rabbit polyclonal POSTN (Abcam, ab 14041) and rabbit monoclonal phospho-STAT1 (Tyr701; Cell Signaling) were used.

    Techniques: Expressing, Activation Assay, Immunohistochemistry, In Vivo, Injection, Stable Transfection, Transfection, shRNA, Western Blot, Isolation, Transduction

    5′ppp-RNA activation of RIG-I stimulates the IRF3–IFNβ–IFNAR–STAT1–IFIT1 pathway to induce cell death of A549 cells. Cells were transfected with Lyovec control or 1 μg/ml of 5′ppp-RNA with Lyovec. b , d , f Lysates were collected at indicated times after 5′ppp-RNA transfection and assayed for the indicated proteins. Blots are representative of three independent experiments. a , c , e , g Cell viability was determined using Cell Titer assay and normalized to the Lyovec control after 72 h. Specifically, cells were a treated with human recombinant IFNβ for the stated times, b , c pre-and cotreated with 100 or 200 μM Amlexanox, a TBK1 antagonist 1 h prior to 5′ppp-RNA transfection, d , e pre-and co-treated with 0.1 μg/ml B18R an IFNAR antagonist 1 h prior to 5′ppp-RNA transfection, and f , g transfected with negative siCTRL and siIFIT1 16 h post-transfection of 5′ppp-RNA. Data is represented as mean ± SEM of n = 3 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: Annexin-A1 promotes RIG-I-dependent signaling and apoptosis via regulation of the IRF3–IFNAR–STAT1–IFIT1 pathway in A549 lung epithelial cells

    doi: 10.1038/s41419-020-2625-7

    Figure Lengend Snippet: 5′ppp-RNA activation of RIG-I stimulates the IRF3–IFNβ–IFNAR–STAT1–IFIT1 pathway to induce cell death of A549 cells. Cells were transfected with Lyovec control or 1 μg/ml of 5′ppp-RNA with Lyovec. b , d , f Lysates were collected at indicated times after 5′ppp-RNA transfection and assayed for the indicated proteins. Blots are representative of three independent experiments. a , c , e , g Cell viability was determined using Cell Titer assay and normalized to the Lyovec control after 72 h. Specifically, cells were a treated with human recombinant IFNβ for the stated times, b , c pre-and cotreated with 100 or 200 μM Amlexanox, a TBK1 antagonist 1 h prior to 5′ppp-RNA transfection, d , e pre-and co-treated with 0.1 μg/ml B18R an IFNAR antagonist 1 h prior to 5′ppp-RNA transfection, and f , g transfected with negative siCTRL and siIFIT1 16 h post-transfection of 5′ppp-RNA. Data is represented as mean ± SEM of n = 3 independent experiments. * P

    Article Snippet: Primary antibodies used were Actin (Santa Cruz, TX, US), IFIT1 (GeneTex, CA, USA), Tubulin (Sigma-Aldrich, MI, USA), Annexin-A1, Cleaved Caspase 3, Cleaved caspase 7, caspase 3, caspase 7, GAPDH, phospho and total IRF3, phospho and total TBK1, RIG-I, phospho, and total STAT1 (Cell signaling Technology, MA, USA).

    Techniques: Activation Assay, Transfection, Titer Assay, Recombinant

    ANXA1 promotes IFNβ production and more rapid IRF3 activation after RIG-I stimulation. Cells were transfected with Lyovec control or 1 μg/ml of 5′ppp-RNA with Lyovec. a – c TNF-α, IL6, and IFNβ expression was measured with quantitative real-time PCR after the indicated times. d IFNβ production was measured using ELISA after 48 h. e Lysates were collected at indicated times after 5′ppp-RNA transfection and assayed for the indicated proteins. Blots are representative of three independent experiments. e , f Phosho-IRF3 or STAT1 was quantified and normalized to their respective total proteins and actin. Data is represented as mean ± SEM of n = 4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: Annexin-A1 promotes RIG-I-dependent signaling and apoptosis via regulation of the IRF3–IFNAR–STAT1–IFIT1 pathway in A549 lung epithelial cells

    doi: 10.1038/s41419-020-2625-7

    Figure Lengend Snippet: ANXA1 promotes IFNβ production and more rapid IRF3 activation after RIG-I stimulation. Cells were transfected with Lyovec control or 1 μg/ml of 5′ppp-RNA with Lyovec. a – c TNF-α, IL6, and IFNβ expression was measured with quantitative real-time PCR after the indicated times. d IFNβ production was measured using ELISA after 48 h. e Lysates were collected at indicated times after 5′ppp-RNA transfection and assayed for the indicated proteins. Blots are representative of three independent experiments. e , f Phosho-IRF3 or STAT1 was quantified and normalized to their respective total proteins and actin. Data is represented as mean ± SEM of n = 4 independent experiments. * P

    Article Snippet: Primary antibodies used were Actin (Santa Cruz, TX, US), IFIT1 (GeneTex, CA, USA), Tubulin (Sigma-Aldrich, MI, USA), Annexin-A1, Cleaved Caspase 3, Cleaved caspase 7, caspase 3, caspase 7, GAPDH, phospho and total IRF3, phospho and total TBK1, RIG-I, phospho, and total STAT1 (Cell signaling Technology, MA, USA).

    Techniques: Activation Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Effect of IL-26 on STAT1, STAT3, and STAT6 activation in M1 and M2 macrophage differentiation. ( A ) RAW264.7 cells were serum-starved for 16 h and treated with IFN-γ (20 ng/mL) or IL-4 (20 ng/mL) in the presence or absence of IL-26, and ( B ) THP-1 cells were treated with IFN-γ (20 ng/mL) plus LPS (10 ng/mL) or IL-4 (20 ng/mL) plus IL-13 (20 ng/mL) in the presence or absence of IL-26 for 2 and 4 h to further detect phosphorylated or non-phosphorylated STAT1, STAT3, and STAT6 proteins. Cell extracts were analyzed by western blot using antibodies specifically directed against the phosphorylated forms of STATs, compared with data obtained with antibodies directed against the unphosphorylated states of the kinases. Equal amounts of protein were loaded in each lane as demonstrated by the level of GAPDH. A representative result of at least three independent experiments is shown. (FL+IL-26: IFN-γ+LPS+IL-26; 413+IL-26: IL-4 + IL-13+IL-26)

    Journal: Cells

    Article Title: Interleukin 26 Skews Macrophage Polarization Towards M1 Phenotype by Activating cJUN and the NF-κB Pathway

    doi: 10.3390/cells9040938

    Figure Lengend Snippet: Effect of IL-26 on STAT1, STAT3, and STAT6 activation in M1 and M2 macrophage differentiation. ( A ) RAW264.7 cells were serum-starved for 16 h and treated with IFN-γ (20 ng/mL) or IL-4 (20 ng/mL) in the presence or absence of IL-26, and ( B ) THP-1 cells were treated with IFN-γ (20 ng/mL) plus LPS (10 ng/mL) or IL-4 (20 ng/mL) plus IL-13 (20 ng/mL) in the presence or absence of IL-26 for 2 and 4 h to further detect phosphorylated or non-phosphorylated STAT1, STAT3, and STAT6 proteins. Cell extracts were analyzed by western blot using antibodies specifically directed against the phosphorylated forms of STATs, compared with data obtained with antibodies directed against the unphosphorylated states of the kinases. Equal amounts of protein were loaded in each lane as demonstrated by the level of GAPDH. A representative result of at least three independent experiments is shown. (FL+IL-26: IFN-γ+LPS+IL-26; 413+IL-26: IL-4 + IL-13+IL-26)

    Article Snippet: Phosphorylated and non-phosphorylated STAT1, STAT3, STAT6, and NF-κB were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Western Blot