phosphorylated stat1  (Cell Signaling Technology Inc)

 
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  • 99
    Name:
    Phospho Stat1 Tyr701 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:
    9171
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr701 of human Stat1. Antibodies are purified by protein A and peptide affinity chromatography.
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc phosphorylated stat1
    MxA expression in human GICs depends on signaling through IFNAR1 and IFNAR2. (A) Basal expression levels of IFNAR1 and IFNAR2 were assessed in LN-18, LN-428, D247MG, LN-319, A172, U87MG, T98G, LN-308 and LN-229, T-325, T-269, ZH-161, ZH-305, or S-24 cells by real-time PCR (left) (median expression levels ± SE are shown from 2 independent experiments). Cell surface IFNAR2 protein was analyzed by flow cytometry (1 out of 2 independent experiments is shown). Isotype control antibody (gray) and specific antibody (black) are shown in the histograms (right). (B) SiRNA-mediated gene silencing of IFNAR1 (siIFNAR1), IFNAR2 (siIFNAR2), or IFNAR1 and IFNAR2 in parallel (siIFNAR1/2) in T-325 or ZH-161 cells was performed by electroporation and confirmed for IFNAR1, IFNAR2, and MxA by real-time PCR 24 h posttransfection (median expression levels ± SE are shown from 3 independent experiments) and for IFNAR2 by flow cytometry at 48 h following transfection. Isotype control antibody (gray) and specific antibody (black) are shown in the histograms. (C) <t>Phospho-STAT1,</t> STAT1, and MxA levels of control, siIFNAR1, siIFNAR2, or double knockdown cells (siIFNAR1/2) were determined 48 h after transfection by immunoblot (1 out of 3 independent experiments is shown).
    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/phosphorylated stat1/product/Cell Signaling Technology Inc
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    phosphorylated stat1 - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Autocrine activation of the IFN signaling pathway may promote immune escape in glioblastoma"

    Article Title: Autocrine activation of the IFN signaling pathway may promote immune escape in glioblastoma

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nox051

    MxA expression in human GICs depends on signaling through IFNAR1 and IFNAR2. (A) Basal expression levels of IFNAR1 and IFNAR2 were assessed in LN-18, LN-428, D247MG, LN-319, A172, U87MG, T98G, LN-308 and LN-229, T-325, T-269, ZH-161, ZH-305, or S-24 cells by real-time PCR (left) (median expression levels ± SE are shown from 2 independent experiments). Cell surface IFNAR2 protein was analyzed by flow cytometry (1 out of 2 independent experiments is shown). Isotype control antibody (gray) and specific antibody (black) are shown in the histograms (right). (B) SiRNA-mediated gene silencing of IFNAR1 (siIFNAR1), IFNAR2 (siIFNAR2), or IFNAR1 and IFNAR2 in parallel (siIFNAR1/2) in T-325 or ZH-161 cells was performed by electroporation and confirmed for IFNAR1, IFNAR2, and MxA by real-time PCR 24 h posttransfection (median expression levels ± SE are shown from 3 independent experiments) and for IFNAR2 by flow cytometry at 48 h following transfection. Isotype control antibody (gray) and specific antibody (black) are shown in the histograms. (C) Phospho-STAT1, STAT1, and MxA levels of control, siIFNAR1, siIFNAR2, or double knockdown cells (siIFNAR1/2) were determined 48 h after transfection by immunoblot (1 out of 3 independent experiments is shown).
    Figure Legend Snippet: MxA expression in human GICs depends on signaling through IFNAR1 and IFNAR2. (A) Basal expression levels of IFNAR1 and IFNAR2 were assessed in LN-18, LN-428, D247MG, LN-319, A172, U87MG, T98G, LN-308 and LN-229, T-325, T-269, ZH-161, ZH-305, or S-24 cells by real-time PCR (left) (median expression levels ± SE are shown from 2 independent experiments). Cell surface IFNAR2 protein was analyzed by flow cytometry (1 out of 2 independent experiments is shown). Isotype control antibody (gray) and specific antibody (black) are shown in the histograms (right). (B) SiRNA-mediated gene silencing of IFNAR1 (siIFNAR1), IFNAR2 (siIFNAR2), or IFNAR1 and IFNAR2 in parallel (siIFNAR1/2) in T-325 or ZH-161 cells was performed by electroporation and confirmed for IFNAR1, IFNAR2, and MxA by real-time PCR 24 h posttransfection (median expression levels ± SE are shown from 3 independent experiments) and for IFNAR2 by flow cytometry at 48 h following transfection. Isotype control antibody (gray) and specific antibody (black) are shown in the histograms. (C) Phospho-STAT1, STAT1, and MxA levels of control, siIFNAR1, siIFNAR2, or double knockdown cells (siIFNAR1/2) were determined 48 h after transfection by immunoblot (1 out of 3 independent experiments is shown).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Electroporation, Transfection

    MxA is expressed in gliomas in vivo. (A) MxA mRNA expression levels in gliomas of different WHO grades were analyzed using data from the database of TCGA (left). Overall survival analysis within the TCGA database for glioblastoma patients with high versus low MxA expression was performed by Kaplan–Meier analysis. The median was used as cutoff (right). (B) MxA protein levels were assessed by immunohistochemistry on a glioma tissue microarray and quantified by H scoring (left). Phospho-STAT1 protein levels were analyzed by immunohistochemistry on a TMA and quantified by H scoring. A correlation analysis of pSTAT1 H scores with MxA H scores is shown (right). (C) Representative images of normal brain and glioblastoma specimens with low, intermediate, and high MxA levels are shown (scale bar, 100 µm or 10 µm for 20x or 40x magnification, respectively). (D) MxA/CD45 costaining was performed on a glioma TMA and the number of double-positive cells was counted.
    Figure Legend Snippet: MxA is expressed in gliomas in vivo. (A) MxA mRNA expression levels in gliomas of different WHO grades were analyzed using data from the database of TCGA (left). Overall survival analysis within the TCGA database for glioblastoma patients with high versus low MxA expression was performed by Kaplan–Meier analysis. The median was used as cutoff (right). (B) MxA protein levels were assessed by immunohistochemistry on a glioma tissue microarray and quantified by H scoring (left). Phospho-STAT1 protein levels were analyzed by immunohistochemistry on a TMA and quantified by H scoring. A correlation analysis of pSTAT1 H scores with MxA H scores is shown (right). (C) Representative images of normal brain and glioblastoma specimens with low, intermediate, and high MxA levels are shown (scale bar, 100 µm or 10 µm for 20x or 40x magnification, respectively). (D) MxA/CD45 costaining was performed on a glioma TMA and the number of double-positive cells was counted.

    Techniques Used: In Vivo, Expressing, Immunohistochemistry, Microarray

    IFN-β target genes are expressed in glioma cells. (A) The human LTC LN-18, LN-428, D247MG, LN-319, A172, U87MG, T98G, LN-308 and LN-229, and the GICs T-325, T-269, ZH-161, ZH-305, and S-24 were cultured without or with IFN-β (150 IU/mL) for 48 h and MxA mRNA expression levels were determined using real-time PCR (median expression levels ± SE are shown from 3 independent experiments). (B) The cells were treated as in (A). Whole cell lysates were subsequently analyzed for pSTAT1, STAT1, and MxA protein levels by immunoblot using actin as a loading control (1 out of 2 independent experiments is shown). (C) LN-308 or ZH-161 cells, untreated or exposed to IFN-β (150 IU/mL) for 48 h, were analyzed for MxA protein levels by immunofluorescence (scale bar, 50 µm). (D) LN-308, LN-229, T-325, or ZH-161 cells were cultured in various medium conditions as indicated for 48 h (SF, serum-free DMEM; NB, Neurobasal medium). Whole cell lysates were assessed for MxA expression levels using immunoblot. Actin was used as loading control.
    Figure Legend Snippet: IFN-β target genes are expressed in glioma cells. (A) The human LTC LN-18, LN-428, D247MG, LN-319, A172, U87MG, T98G, LN-308 and LN-229, and the GICs T-325, T-269, ZH-161, ZH-305, and S-24 were cultured without or with IFN-β (150 IU/mL) for 48 h and MxA mRNA expression levels were determined using real-time PCR (median expression levels ± SE are shown from 3 independent experiments). (B) The cells were treated as in (A). Whole cell lysates were subsequently analyzed for pSTAT1, STAT1, and MxA protein levels by immunoblot using actin as a loading control (1 out of 2 independent experiments is shown). (C) LN-308 or ZH-161 cells, untreated or exposed to IFN-β (150 IU/mL) for 48 h, were analyzed for MxA protein levels by immunofluorescence (scale bar, 50 µm). (D) LN-308, LN-229, T-325, or ZH-161 cells were cultured in various medium conditions as indicated for 48 h (SF, serum-free DMEM; NB, Neurobasal medium). Whole cell lysates were assessed for MxA expression levels using immunoblot. Actin was used as loading control.

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence

    Glioma-derived IFN-α or IFN-β induces autocrine signaling. (A) IFN-α or IFN-β mRNA expression levels were determined in LN-308, LN-229, T-325, T-269, ZH-161, ZH-305, or S-24 cells using real-time PCR (median expression levels ± SE are shown from 2 independent experiments). (B, C) SiRNA-mediated gene silencing of IFN-α (si_IFN-α) or IFN-β (si_IFN-β) was performed using electroporation in T-325 or ZH-161 cells. MxA mRNA expression was determined 24 h posttransfection by real-time PCR (median expression levels ± SE are shown from 2 independent experiments) (B), while pSTAT1, STAT1, and MxA protein levels were assessed at 48 h following transfection by immunoblot. Actin was used as a loading control (1 out of 3 independent experiments is shown) (C).
    Figure Legend Snippet: Glioma-derived IFN-α or IFN-β induces autocrine signaling. (A) IFN-α or IFN-β mRNA expression levels were determined in LN-308, LN-229, T-325, T-269, ZH-161, ZH-305, or S-24 cells using real-time PCR (median expression levels ± SE are shown from 2 independent experiments). (B, C) SiRNA-mediated gene silencing of IFN-α (si_IFN-α) or IFN-β (si_IFN-β) was performed using electroporation in T-325 or ZH-161 cells. MxA mRNA expression was determined 24 h posttransfection by real-time PCR (median expression levels ± SE are shown from 2 independent experiments) (B), while pSTAT1, STAT1, and MxA protein levels were assessed at 48 h following transfection by immunoblot. Actin was used as a loading control (1 out of 3 independent experiments is shown) (C).

    Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Electroporation, Transfection

    2) Product Images from "JAK-STAT signaling pathways are activated in the brain following reovirus infection"

    Article Title: JAK-STAT signaling pathways are activated in the brain following reovirus infection

    Journal:

    doi: 10.1080/13550280701344983

    Pretreatment with interferon-specific antibodies has differing effects on T3A-induced STAT1 phosphorylation. Cortical neuron cultures were mock or T3A infected (MOI of 100) following 4 h pretreatment with antibody directed against IFN- γ (10 μg/ml)
    Figure Legend Snippet: Pretreatment with interferon-specific antibodies has differing effects on T3A-induced STAT1 phosphorylation. Cortical neuron cultures were mock or T3A infected (MOI of 100) following 4 h pretreatment with antibody directed against IFN- γ (10 μg/ml)

    Techniques Used: Infection

    Reovirus localizes to similar brain regions in STAT1 gene-deficient mice. Newborn STAT1 gene-deficient mouse pups and syngeneic controls (129SvEv) were T3A or T1L infected at 2 days of age with 100 PFU of virus. Brain injury was evaluated in H E-stained
    Figure Legend Snippet: Reovirus localizes to similar brain regions in STAT1 gene-deficient mice. Newborn STAT1 gene-deficient mouse pups and syngeneic controls (129SvEv) were T3A or T1L infected at 2 days of age with 100 PFU of virus. Brain injury was evaluated in H E-stained

    Techniques Used: Mouse Assay, Infection, Staining

    T3A induces STAT1 phosphorylation in the brain of neonatal mice following intracerebral inoculation. Two-day-old Swiss Webster mice were mock or T3A infected (1 × 10 3 PFU, i.c.) and sacrificed at 4 or 8 days post infection. Whole-brain lysates
    Figure Legend Snippet: T3A induces STAT1 phosphorylation in the brain of neonatal mice following intracerebral inoculation. Two-day-old Swiss Webster mice were mock or T3A infected (1 × 10 3 PFU, i.c.) and sacrificed at 4 or 8 days post infection. Whole-brain lysates

    Techniques Used: Mouse Assay, Infection

    Y701-phosphorylated STAT1 is expressed in areas surrounding brain injury following T3A infection. Two-day-old Swiss Webster mice were mock or T3A infected (i.c. inoculation of 1 × 10 3 PFU) and sacrificed at 8 days post infection. pY701-STAT1 (fluorescein)
    Figure Legend Snippet: Y701-phosphorylated STAT1 is expressed in areas surrounding brain injury following T3A infection. Two-day-old Swiss Webster mice were mock or T3A infected (i.c. inoculation of 1 × 10 3 PFU) and sacrificed at 8 days post infection. pY701-STAT1 (fluorescein)

    Techniques Used: Infection, Mouse Assay

    Phosphorylation of STAT1 at Y701 is mediated via JAK activation. Whole-brain lysates were prepared from mock- and T3A-infected (1 × 10 3 PFU, i.c.) mice at 8 days post infection and probed, by Western blot, for activated JAK2 (using an antibody
    Figure Legend Snippet: Phosphorylation of STAT1 at Y701 is mediated via JAK activation. Whole-brain lysates were prepared from mock- and T3A-infected (1 × 10 3 PFU, i.c.) mice at 8 days post infection and probed, by Western blot, for activated JAK2 (using an antibody

    Techniques Used: Activation Assay, Infection, Mouse Assay, Western Blot

    Mice lacking the STAT1 gene suffer accelerated mortality and increased viral brain titers. Newborn STAT1 gene-deficient mouse pups ( dashed lines ) and syngeneic controls (129SvEv) ( solid lines ) were transferred to Swiss Webster surrogate mothers and T3A
    Figure Legend Snippet: Mice lacking the STAT1 gene suffer accelerated mortality and increased viral brain titers. Newborn STAT1 gene-deficient mouse pups ( dashed lines ) and syngeneic controls (129SvEv) ( solid lines ) were transferred to Swiss Webster surrogate mothers and T3A

    Techniques Used: Mouse Assay

    Reovirus induces STAT1 phosphorylation at Y701 in primary neuronal cultures following infection. Cortical neuron cultures were mock or reovirus infected (MOI of 100). Dual-label immunocytochemical staining was performed to identify reovirus antigen σ
    Figure Legend Snippet: Reovirus induces STAT1 phosphorylation at Y701 in primary neuronal cultures following infection. Cortical neuron cultures were mock or reovirus infected (MOI of 100). Dual-label immunocytochemical staining was performed to identify reovirus antigen σ

    Techniques Used: Infection, Staining

    3) Product Images from "Interdependent and independent roles of type I interferons and IL-6 in innate immune, neuroinflammatory and sickness behaviour responses to systemic poly I:C"

    Article Title: Interdependent and independent roles of type I interferons and IL-6 in innate immune, neuroinflammatory and sickness behaviour responses to systemic poly I:C

    Journal: Brain, Behavior, and Immunity

    doi: 10.1016/j.bbi.2015.04.009

    Expression of total and phosphorylated STAT1 in WT and IFNAR1 −/− mice. Western blot of basal STAT1 in WT and IFNAR1 −/− hippocampal homogenates (a). Western blot analysis of STAT1 and phosphorylated STAT1 in WT and IFNAR1 −/− mice post-poly I:C challenge at 1 and 3 h (b). Quantitative histogram of basal STAT1 in WT and IFNAR1 −/− mice and levels of STAT1 at 1 and 3 h post-poly I:C (c). Significant differences by Students t test are denoted by ∗∗ p = 0.0015. Expression of stat1 mRNA (d). Data are expressed as mean ± SEM and analysed by two-way ANOVA with treatment and strain as factors; n = 5 for WT + saline, n = 4 for WT + pI:C 1 h, n = 5 for WT + pI:C 3 h and n = 5 for IFNAR1 −/− + saline, n = 4 for IFNAR1 −/− + pI:C 1 h, n = 5 for IFNAR1 −/− + pI:C 3 h. Significant main effect of treatment by Bonferroni post-hoc is denoted by ∗∗∗ p
    Figure Legend Snippet: Expression of total and phosphorylated STAT1 in WT and IFNAR1 −/− mice. Western blot of basal STAT1 in WT and IFNAR1 −/− hippocampal homogenates (a). Western blot analysis of STAT1 and phosphorylated STAT1 in WT and IFNAR1 −/− mice post-poly I:C challenge at 1 and 3 h (b). Quantitative histogram of basal STAT1 in WT and IFNAR1 −/− mice and levels of STAT1 at 1 and 3 h post-poly I:C (c). Significant differences by Students t test are denoted by ∗∗ p = 0.0015. Expression of stat1 mRNA (d). Data are expressed as mean ± SEM and analysed by two-way ANOVA with treatment and strain as factors; n = 5 for WT + saline, n = 4 for WT + pI:C 1 h, n = 5 for WT + pI:C 3 h and n = 5 for IFNAR1 −/− + saline, n = 4 for IFNAR1 −/− + pI:C 1 h, n = 5 for IFNAR1 −/− + pI:C 3 h. Significant main effect of treatment by Bonferroni post-hoc is denoted by ∗∗∗ p

    Techniques Used: Expressing, Mouse Assay, Western Blot

    4) Product Images from "Leukotriene B4 Receptor-2 Promotes Invasiveness and Metastasis of Ovarian Cancer Cells through Signal Transducer and Activator of Transcription 3 (STAT3)-dependent Up-regulation of Matrix Metalloproteinase 2 *"

    Article Title: Leukotriene B4 Receptor-2 Promotes Invasiveness and Metastasis of Ovarian Cancer Cells through Signal Transducer and Activator of Transcription 3 (STAT3)-dependent Up-regulation of Matrix Metalloproteinase 2 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.317131

    STAT3 functions downstream of BLT2-NOX4-ROS signaling to mediate up-regulation of MMP2. A , immunoblot analysis of Tyr 705 -phosphorylated ( Y705 ), Ser 727 -phosphorylated ( S727 ) forms of STAT3 ( p-STAT3 ) or Tyr 701 -phosphorylated ( Y701 ) forms of STAT1 ( p-STAT1
    Figure Legend Snippet: STAT3 functions downstream of BLT2-NOX4-ROS signaling to mediate up-regulation of MMP2. A , immunoblot analysis of Tyr 705 -phosphorylated ( Y705 ), Ser 727 -phosphorylated ( S727 ) forms of STAT3 ( p-STAT3 ) or Tyr 701 -phosphorylated ( Y701 ) forms of STAT1 ( p-STAT1

    Techniques Used:

    5) Product Images from "IL-10 regulates adult neurogenesis by modulating ERK and STAT3 activity"

    Article Title: IL-10 regulates adult neurogenesis by modulating ERK and STAT3 activity

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2015.00057

    Interleukin-10 increases serine STAT3 phosphorylation in Nestin+ SVZ progenitors. (A) Phosphorylation of STAT3 Ser727 was induced from 5 min after IL-10 (50 ng/mL) addition in primary cultures of the SVZ. Stimulation of pSTAT3 Tyr701 , STAT1, and JAK1 and JAK2 were not observed ( n = 6 per time point). (B) Double-immunofluorescences stainings show that STAT3 Ser727 immunoreactivity (green) was increased in Nestin+ progenitors (red) in IL-10 stimulated cultures (30 min after administration), compared with vehicle treatment (control). TO-PRO (blue) labeled all nuclei. ( n = 5). (C) Histogram represents the nuclear immunoreactivity intensity of STAT3 Ser727 per Nestin+ cells in control and after IL-10 stimulation. After IL-10 stimulation phosphorylation of STAT3 Ser727 was exacerbated in Nestin+ progenitors ( n = 4). Scale bar (B) , 30 μm. Data are represented as mean ± SEM. * P ≤ 0.05.
    Figure Legend Snippet: Interleukin-10 increases serine STAT3 phosphorylation in Nestin+ SVZ progenitors. (A) Phosphorylation of STAT3 Ser727 was induced from 5 min after IL-10 (50 ng/mL) addition in primary cultures of the SVZ. Stimulation of pSTAT3 Tyr701 , STAT1, and JAK1 and JAK2 were not observed ( n = 6 per time point). (B) Double-immunofluorescences stainings show that STAT3 Ser727 immunoreactivity (green) was increased in Nestin+ progenitors (red) in IL-10 stimulated cultures (30 min after administration), compared with vehicle treatment (control). TO-PRO (blue) labeled all nuclei. ( n = 5). (C) Histogram represents the nuclear immunoreactivity intensity of STAT3 Ser727 per Nestin+ cells in control and after IL-10 stimulation. After IL-10 stimulation phosphorylation of STAT3 Ser727 was exacerbated in Nestin+ progenitors ( n = 4). Scale bar (B) , 30 μm. Data are represented as mean ± SEM. * P ≤ 0.05.

    Techniques Used: Labeling

    6) Product Images from "The molecular basis of IL-21-mediated proliferation"

    Article Title: The molecular basis of IL-21-mediated proliferation

    Journal:

    doi: 10.1182/blood-2006-10-054973

    Stat1/Stat3-deficient CD8 + T cells exhibit diminished expansion in response to IL-15 + IL-21 . (A) Splenic CD8 + T cells were isolated from control floxed Stat3 mice or Stat1 −/− Stat3 −/− mice and then Western blotted as in
    Figure Legend Snippet: Stat1/Stat3-deficient CD8 + T cells exhibit diminished expansion in response to IL-15 + IL-21 . (A) Splenic CD8 + T cells were isolated from control floxed Stat3 mice or Stat1 −/− Stat3 −/− mice and then Western blotted as in

    Techniques Used: Isolation, Mouse Assay, Western Blot

    IL-21–induced Stat1 and Stat3 activation requires Y510 . (A) Ba/F3 cells stably transfected with the indicated constructs were cultured in IL-3–free medium for 5 hours and treated with 20 ng/mL IL-21 for 0, 5, or 30 minutes. Cells were
    Figure Legend Snippet: IL-21–induced Stat1 and Stat3 activation requires Y510 . (A) Ba/F3 cells stably transfected with the indicated constructs were cultured in IL-3–free medium for 5 hours and treated with 20 ng/mL IL-21 for 0, 5, or 30 minutes. Cells were

    Techniques Used: Activation Assay, Stable Transfection, Transfection, Construct, Cell Culture

    Stat1, Stat3, and Stat5 are differentially activated by IL-21 . (A) WT splenocytes were preactivated for 2 days with 2 ng/mL PMA + 1 μg/mL ionomycin, expanded with 10 U/mL IL-2 for 1 day, rested overnight in fresh medium without IL-2, and then
    Figure Legend Snippet: Stat1, Stat3, and Stat5 are differentially activated by IL-21 . (A) WT splenocytes were preactivated for 2 days with 2 ng/mL PMA + 1 μg/mL ionomycin, expanded with 10 U/mL IL-2 for 1 day, rested overnight in fresh medium without IL-2, and then

    Techniques Used:

    7) Product Images from "A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling"

    Article Title: A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling

    Journal: Molecular carcinogenesis

    doi: 10.1002/mc.22132

    Impact of Stat1 deficiency on Cox-2 induction following treatment with TPA and CHRY
    Figure Legend Snippet: Impact of Stat1 deficiency on Cox-2 induction following treatment with TPA and CHRY

    Techniques Used:

    Impact of Stat1 deficiency on skin tumor promotion
    Figure Legend Snippet: Impact of Stat1 deficiency on skin tumor promotion

    Techniques Used:

    Analysis of Stat1 phosphorylation and protein level following tumor promoter treatment
    Figure Legend Snippet: Analysis of Stat1 phosphorylation and protein level following tumor promoter treatment

    Techniques Used:

    8) Product Images from "High salt diet accelerates the progression of murine lupus through dendritic cells via the p38 MAPK and STAT1 signaling pathways"

    Article Title: High salt diet accelerates the progression of murine lupus through dendritic cells via the p38 MAPK and STAT1 signaling pathways

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-0139-5

    NaCl facilitated the function of dendritic cells through the p38 MAPK–STAT1 pathway. Bone morrow-derived dendritic cells were incubated with ALD-DNA with or without NaCl (20 mM) and were treated with STAT1 inhibitor (2 μg/ml), p38 MAPK inhibitor (5 μM), or control (DMSO). a Quantitative RT-PCR array showing the control-treated BMDCs and NaCl-treated BMDCs; C1, C2, or C3 represents control-treated BMDCs and N1, N2, or N3 represents NaCl-treated BMDCs. STAT1 expression was the only parameter to show a statistically significant difference ( p value = 0.0034). b Total STAT1, p38 MAPK, phosphorylated STAT1, and phosphorylated p38 MAPK in control-treated BMDCs and NaCl-treated BMDCs. c Flow cytometry analysis showing the activation and maturation markers in BMDCs with or without NaCl treatment in the presence or absence of the STAT1 inhibitor. d Flow cytometry analysis showing IFN-γ and TNF-α expression in BMDCs with or without NaCl treatment in the presence or absence of the STAT1 inhibitor. e Bone marrow-derived dendritic cells from C57BL/6 mice were pretreated with 10 ng/ml LPS and/or 20 mM NaCl and/or 2 μg/ml STAT1 inhibitor and/or 5 μM p38 MAPK inhibitor for 24 h and then were collected for coculture with CFSE-labeled T cells from Balb/c mice for 72 h; the CFSE in T cells was analyzed. f Bone marrow-derived dendritic cells were incubated ALD-DNA with or without NaCl treatment (20 mM) in the presence or absence of 2 μg/ml STAT1 inhibitor and were then transferred to normal C57BL/6 mice ( n = 5 in each experiment). ELISA results showing semiquantitative measurement of IgG, IgG1, IgG2a, and IgG2b anti-dsDNA in sera from each group of lupus mice 4 weeks after BMDC-ALD-DNA injection. g Bone marrow-derived dendritic cells from wild type and STAT1 −/− mice were incubated with ALD-DNA with or without NaCl treatment (20 mM) were then transferred to normal C57BL/6 mice ( n = 5 in each individual experiment). ELISA measurements showing IgG, IgG1, IgG2a, and IgG2b anti-dsDNA in sera from each group of lupus mice 8 days after BMDC-ALD-DNA injection. The results are presented as the mean ± s.e.m. from three independent experiments. ns indicates no significance, ** p
    Figure Legend Snippet: NaCl facilitated the function of dendritic cells through the p38 MAPK–STAT1 pathway. Bone morrow-derived dendritic cells were incubated with ALD-DNA with or without NaCl (20 mM) and were treated with STAT1 inhibitor (2 μg/ml), p38 MAPK inhibitor (5 μM), or control (DMSO). a Quantitative RT-PCR array showing the control-treated BMDCs and NaCl-treated BMDCs; C1, C2, or C3 represents control-treated BMDCs and N1, N2, or N3 represents NaCl-treated BMDCs. STAT1 expression was the only parameter to show a statistically significant difference ( p value = 0.0034). b Total STAT1, p38 MAPK, phosphorylated STAT1, and phosphorylated p38 MAPK in control-treated BMDCs and NaCl-treated BMDCs. c Flow cytometry analysis showing the activation and maturation markers in BMDCs with or without NaCl treatment in the presence or absence of the STAT1 inhibitor. d Flow cytometry analysis showing IFN-γ and TNF-α expression in BMDCs with or without NaCl treatment in the presence or absence of the STAT1 inhibitor. e Bone marrow-derived dendritic cells from C57BL/6 mice were pretreated with 10 ng/ml LPS and/or 20 mM NaCl and/or 2 μg/ml STAT1 inhibitor and/or 5 μM p38 MAPK inhibitor for 24 h and then were collected for coculture with CFSE-labeled T cells from Balb/c mice for 72 h; the CFSE in T cells was analyzed. f Bone marrow-derived dendritic cells were incubated ALD-DNA with or without NaCl treatment (20 mM) in the presence or absence of 2 μg/ml STAT1 inhibitor and were then transferred to normal C57BL/6 mice ( n = 5 in each experiment). ELISA results showing semiquantitative measurement of IgG, IgG1, IgG2a, and IgG2b anti-dsDNA in sera from each group of lupus mice 4 weeks after BMDC-ALD-DNA injection. g Bone marrow-derived dendritic cells from wild type and STAT1 −/− mice were incubated with ALD-DNA with or without NaCl treatment (20 mM) were then transferred to normal C57BL/6 mice ( n = 5 in each individual experiment). ELISA measurements showing IgG, IgG1, IgG2a, and IgG2b anti-dsDNA in sera from each group of lupus mice 8 days after BMDC-ALD-DNA injection. The results are presented as the mean ± s.e.m. from three independent experiments. ns indicates no significance, ** p

    Techniques Used: Derivative Assay, Incubation, Quantitative RT-PCR, Expressing, Flow Cytometry, Activation Assay, Mouse Assay, Labeling, Enzyme-linked Immunosorbent Assay, Injection

    9) Product Images from "Laquinimod, a Quinoline-3-Carboxamide, Induces Type II Myeloid Cells That Modulate Central Nervous System Autoimmunity"

    Article Title: Laquinimod, a Quinoline-3-Carboxamide, Induces Type II Myeloid Cells That Modulate Central Nervous System Autoimmunity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033797

    Laquinimod-induced type II (M2) monocytes reverse established EAE. Laquinimod-induced anti-inflammatory cytokine shift in CD11b + CD11c − and CD11c + cells. (A) FACS analysis of intracellular production of TNF, IL12/23p40, IL-6 and IL-10 by CD11b + CD11c − and CD11c + cells isolated from spleens of naive (unimmunized) mice treated with laquinimod or vehicle. (B) Cell surface FACS analysis of MHC II, proinflammatory and inhibitory costimulatory molecules on CD11b + CD11c − cells. (C) In vivo laquinimod treatment affects signaling pathways that participate in proinflammatory cytokine production. Protein extracts were isolated from peritoneal macrophages of naïve C57BL/6 mice treated with laquinimod or vehicle and stimulated with LPS for various time points. Phosphorylated (P) STAT1, (P) p38-MAPK, Pan-STAT1 and Pan-p38-MAPK were detected by Western blot analysis. (D) CD11b + cells from laquinimod-treated donor mice reversed established EAE. 5×10 6 purified splenic CD11b + cells from mice treated with laquinimod or vehicle were injected i.v. into recipient C57BL/6 mice immunized with MOG p35–55 after they developed a disease grade of 2 (black arrow indicates time point of adoptive transfer, (n = 5/group). (E) Quantification showed reduced total number of inflammatory foci after adoptive transfer of in vivo laquinimod treated CD11b + cells into C57BL/6 mice immunized with MOG p35-55. Data shown in panels above are representative of three independent experiments. For EAE disease course, mean disease score ± s.e.m. are displayed; *P
    Figure Legend Snippet: Laquinimod-induced type II (M2) monocytes reverse established EAE. Laquinimod-induced anti-inflammatory cytokine shift in CD11b + CD11c − and CD11c + cells. (A) FACS analysis of intracellular production of TNF, IL12/23p40, IL-6 and IL-10 by CD11b + CD11c − and CD11c + cells isolated from spleens of naive (unimmunized) mice treated with laquinimod or vehicle. (B) Cell surface FACS analysis of MHC II, proinflammatory and inhibitory costimulatory molecules on CD11b + CD11c − cells. (C) In vivo laquinimod treatment affects signaling pathways that participate in proinflammatory cytokine production. Protein extracts were isolated from peritoneal macrophages of naïve C57BL/6 mice treated with laquinimod or vehicle and stimulated with LPS for various time points. Phosphorylated (P) STAT1, (P) p38-MAPK, Pan-STAT1 and Pan-p38-MAPK were detected by Western blot analysis. (D) CD11b + cells from laquinimod-treated donor mice reversed established EAE. 5×10 6 purified splenic CD11b + cells from mice treated with laquinimod or vehicle were injected i.v. into recipient C57BL/6 mice immunized with MOG p35–55 after they developed a disease grade of 2 (black arrow indicates time point of adoptive transfer, (n = 5/group). (E) Quantification showed reduced total number of inflammatory foci after adoptive transfer of in vivo laquinimod treated CD11b + cells into C57BL/6 mice immunized with MOG p35-55. Data shown in panels above are representative of three independent experiments. For EAE disease course, mean disease score ± s.e.m. are displayed; *P

    Techniques Used: FACS, Isolation, Mouse Assay, In Vivo, Western Blot, Purification, Injection, Adoptive Transfer Assay

    10) Product Images from "PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation"

    Article Title: PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

    Journal: Nature Communications

    doi: 10.1038/ncomms12849

    The molecular functions of PARP9 and PARP14 in macrophages in vitro . ( a ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulated (TNFα, IL-1β and CCL2/MCP-1) and IL-4 stimulated (MRC1) gene expression in human primary macrophages ( n =8). ( b ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulation (TNFα and iNOS) and IL-4 stimulation (Arg1 and MRC1) gene expression in mouse bone marrow-derived macrophages ( n =3). ( c ) The ratio of phosphorylated STAT1 and STAT6 protein levels to total STAT1 and STAT6 (p STAT1/tSTAT1 ratio and pSTAT6/tSTAT6 ratio) in human primary macrophages ( n =6 and n =5, respectively) of the PARP9 and PARP14 silencing experiments. * P
    Figure Legend Snippet: The molecular functions of PARP9 and PARP14 in macrophages in vitro . ( a ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulated (TNFα, IL-1β and CCL2/MCP-1) and IL-4 stimulated (MRC1) gene expression in human primary macrophages ( n =8). ( b ) The consequences of PARP9 and PARP14 silencing on IFNγ stimulation (TNFα and iNOS) and IL-4 stimulation (Arg1 and MRC1) gene expression in mouse bone marrow-derived macrophages ( n =3). ( c ) The ratio of phosphorylated STAT1 and STAT6 protein levels to total STAT1 and STAT6 (p STAT1/tSTAT1 ratio and pSTAT6/tSTAT6 ratio) in human primary macrophages ( n =6 and n =5, respectively) of the PARP9 and PARP14 silencing experiments. * P

    Techniques Used: In Vitro, Expressing, Derivative Assay

    Identification of PARP14-induced ribosylation sites in STAT1. ( a ) The amino-acid sequence of human STAT1α C terminus. Green amino acids indicate ribosylated peptides; confirmed ribosylation sites are underlined. STAT1 is phosphorylated at indicated tyrosine (red). ( b ; Left panels) MS/MS spectra for the mono-ADP-ribosylated peptides and corresponding unmodified forms. ADP-ribose fragments are annotated in green. *, ribosylation site; m, oxidized methionine. The grey circles indicate background or undetermined ions. (Right panels) MS1-based quantification of PARP9 inhibition of PARP14-mediated STAT1α ribosylation at E657 (upper panel) and E705 (lower panel), respectively. ( c ) Effects of mutated amino acids at E657 and E705 in STAT1 (ribosylation sites for PARP14) on its Tyr701 phosphorylation and pro-inflammatory gene expression in mouse bone marrow-derived macrophages ( n =4). * P
    Figure Legend Snippet: Identification of PARP14-induced ribosylation sites in STAT1. ( a ) The amino-acid sequence of human STAT1α C terminus. Green amino acids indicate ribosylated peptides; confirmed ribosylation sites are underlined. STAT1 is phosphorylated at indicated tyrosine (red). ( b ; Left panels) MS/MS spectra for the mono-ADP-ribosylated peptides and corresponding unmodified forms. ADP-ribose fragments are annotated in green. *, ribosylation site; m, oxidized methionine. The grey circles indicate background or undetermined ions. (Right panels) MS1-based quantification of PARP9 inhibition of PARP14-mediated STAT1α ribosylation at E657 (upper panel) and E705 (lower panel), respectively. ( c ) Effects of mutated amino acids at E657 and E705 in STAT1 (ribosylation sites for PARP14) on its Tyr701 phosphorylation and pro-inflammatory gene expression in mouse bone marrow-derived macrophages ( n =4). * P

    Techniques Used: Sequencing, Mass Spectrometry, Inhibition, Expressing, Derivative Assay

    Role of haematopoietic PARP14 in acute arterial lesion formation in mice. ( a–c ) Cultured peritoneal macrophages derived from PARP14 −/− and PARP14 +/+ mice. ( a ) IFNγ and IL-4 pathway gene expression profiles ( n =3). ( b ) Secretion of inflammatory factors into culture media ( n =3). ( c ) Western blot and corresponding densitometry quantification of phosphorylated STAT1 and STAT6. Each data point is the average of triplicate samples per donor ( n =3). ( d ) Left: representative images of haematoxylin and eosin (H E; top) and Mac3 (bottom) staining. Scale bars, 100 μm. Right: quantification of lesion formation in mechanically injured femoral arteries of PARP14 −/− and PARP +/+ mice. Mac3 staining represents macrophage accumulation ( n =4–5). ( e ) LCM of the neointima followed by gene expression analysis ( n =4). ( f ) Flow cytometry analysis of splenic CD11b+Ly6G− monocytes after induction of mechanically injured femoral arteries of PARP14 +/+ and PARP14 −/− mice ( n =3). ( g ) Representative H E staining images and quantification of neointima formation in mechanically injured femoral arteries after bone marrow transplantation (BMT) PARP14 +/+→+/+ and PARP14 −/−→+/+ mice ( n =6). Scale bars, 100 μm. * P
    Figure Legend Snippet: Role of haematopoietic PARP14 in acute arterial lesion formation in mice. ( a–c ) Cultured peritoneal macrophages derived from PARP14 −/− and PARP14 +/+ mice. ( a ) IFNγ and IL-4 pathway gene expression profiles ( n =3). ( b ) Secretion of inflammatory factors into culture media ( n =3). ( c ) Western blot and corresponding densitometry quantification of phosphorylated STAT1 and STAT6. Each data point is the average of triplicate samples per donor ( n =3). ( d ) Left: representative images of haematoxylin and eosin (H E; top) and Mac3 (bottom) staining. Scale bars, 100 μm. Right: quantification of lesion formation in mechanically injured femoral arteries of PARP14 −/− and PARP +/+ mice. Mac3 staining represents macrophage accumulation ( n =4–5). ( e ) LCM of the neointima followed by gene expression analysis ( n =4). ( f ) Flow cytometry analysis of splenic CD11b+Ly6G− monocytes after induction of mechanically injured femoral arteries of PARP14 +/+ and PARP14 −/− mice ( n =3). ( g ) Representative H E staining images and quantification of neointima formation in mechanically injured femoral arteries after bone marrow transplantation (BMT) PARP14 +/+→+/+ and PARP14 −/−→+/+ mice ( n =6). Scale bars, 100 μm. * P

    Techniques Used: Mouse Assay, Cell Culture, Derivative Assay, Expressing, Western Blot, Staining, Laser Capture Microdissection, Flow Cytometry, Cytometry, Transplantation Assay

    11) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

    Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113318

    CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
    Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

    Techniques Used: Amplification, Isolation, Genome Wide, Expressing

    IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

    Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

    STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Isolation, Quantitative RT-PCR

    12) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

    Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113318

    CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
    Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

    Techniques Used: Amplification, Isolation, Genome Wide, Expressing

    IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

    Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

    STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Isolation, Quantitative RT-PCR

    13) Product Images from "A Novel Requirement for Janus Kinases as Mediators of Drug Resistance Induced by Fibroblast Growth Factor-2 in Human Cancer Cells"

    Article Title: A Novel Requirement for Janus Kinases as Mediators of Drug Resistance Induced by Fibroblast Growth Factor-2 in Human Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019861

    FGF-2 treatment leads to phosphorylation of TYK2, but not STAT1, STAT3 or STAT5A/B. A. FGF-2 did not induce STAT1, STAT3, or STAT5 phosphorylation. U2OS cells were serum-starved overnight and then stimulated with FGF-2 for the indicated times. Cells treated with IFN-γ (500 IU/ml), IL-6 (200 ng/ml)/sIL-6R (250 ng/ml) and OSM (50 ng/ml) were used as positive controls for activation of STAT1, STAT3 and STAT5, respectively. Proteins were analyzed as before using antibodies against phosphorylated STAT1 (Tyr 701 ), phosphorylated STAT3 (Tyr 705 ), phosphorylated STAT5 (Tyr 694 ) and antibodies that recognize both phosphorylated and unphosphorylated proteins. B. FGF-2 induced the phosphorylation of TYK2 in U2OS cells. Cells were incubated in serum-free media and then treated with FGF-2 (10 ng/ml) for the indicated times. TYK2 was immunoprecipitated and western blotting analysis was performed using antibodies against phosphorylated tyrosines and total TYK2. TYK2 was used as a loading control. C. Total cell lysates used to immunoprecipitate TYK2 and from cells transfected with siTYK2 were separated on a 7.5% SDS-PAGE gel and analyzed by western blot. Membranes were probed for TYK2, pERK1/2-Thr 202/185 /Tyr 204/187 and total ERK1/2. β-actin was used as a loading control. ‘ – min.
    Figure Legend Snippet: FGF-2 treatment leads to phosphorylation of TYK2, but not STAT1, STAT3 or STAT5A/B. A. FGF-2 did not induce STAT1, STAT3, or STAT5 phosphorylation. U2OS cells were serum-starved overnight and then stimulated with FGF-2 for the indicated times. Cells treated with IFN-γ (500 IU/ml), IL-6 (200 ng/ml)/sIL-6R (250 ng/ml) and OSM (50 ng/ml) were used as positive controls for activation of STAT1, STAT3 and STAT5, respectively. Proteins were analyzed as before using antibodies against phosphorylated STAT1 (Tyr 701 ), phosphorylated STAT3 (Tyr 705 ), phosphorylated STAT5 (Tyr 694 ) and antibodies that recognize both phosphorylated and unphosphorylated proteins. B. FGF-2 induced the phosphorylation of TYK2 in U2OS cells. Cells were incubated in serum-free media and then treated with FGF-2 (10 ng/ml) for the indicated times. TYK2 was immunoprecipitated and western blotting analysis was performed using antibodies against phosphorylated tyrosines and total TYK2. TYK2 was used as a loading control. C. Total cell lysates used to immunoprecipitate TYK2 and from cells transfected with siTYK2 were separated on a 7.5% SDS-PAGE gel and analyzed by western blot. Membranes were probed for TYK2, pERK1/2-Thr 202/185 /Tyr 204/187 and total ERK1/2. β-actin was used as a loading control. ‘ – min.

    Techniques Used: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Transfection, SDS Page

    Silencing STAT1, STAT3 or STAT5A/B did not impair the ability of FGF-2 to prevent cisplatin-induced apoptosis in U2OS cells. Cells were transfected with 75 nM of siRNA against STAT1, STAT3 or STAT5A/B. Untransfected cells and cells transfected with non-specific siRNA (NS) were used as controls (c.f. Fig. 3 ). Cells were treated as in Figure 3 . A. Mean±SEM of densitometric values (cleaved PARP) from three experiments are shown. B. Cell cycle profiles of three independent experiments are graphically represented as mean±SEM of sub-G1 populations.
    Figure Legend Snippet: Silencing STAT1, STAT3 or STAT5A/B did not impair the ability of FGF-2 to prevent cisplatin-induced apoptosis in U2OS cells. Cells were transfected with 75 nM of siRNA against STAT1, STAT3 or STAT5A/B. Untransfected cells and cells transfected with non-specific siRNA (NS) were used as controls (c.f. Fig. 3 ). Cells were treated as in Figure 3 . A. Mean±SEM of densitometric values (cleaved PARP) from three experiments are shown. B. Cell cycle profiles of three independent experiments are graphically represented as mean±SEM of sub-G1 populations.

    Techniques Used: Transfection

    14) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

    Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113318

    CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
    Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

    Techniques Used: Amplification, Isolation, Genome Wide, Expressing

    IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

    Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

    STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Isolation, Quantitative RT-PCR

    15) Product Images from "Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells"

    Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079518

    Induction of IFIT3 is STAT-2-dependent. A549 cells were infected by mock or DV for 3, 6, and 24 h and protein levels of both phosphorylated and non-phosphorylated STAT1, STAT2, and STAT3 were analyzed by western blotting (A). Treatment with 1000 units IFN-α was used as a positive control. Expression of IFIT3 in DV-infected A549 cells with knockdown of either STAT2 (B), STAT1 (C) or STAT3 (D) was determined by western blotting (B, C, and D) or quantitative RT/PCR (B). Both shRNA and siRNA were used as the approaches for STAT1/STAT2 and STAT3, respectively, as described in Materials and Methods. Knockdown with shGFP or si-Ctl was used as a negative control. Data show representative results and analysis pooled from at least 3 independent experiments. The analysis was performed by ANOVA as described in Materials and Methods. **P
    Figure Legend Snippet: Induction of IFIT3 is STAT-2-dependent. A549 cells were infected by mock or DV for 3, 6, and 24 h and protein levels of both phosphorylated and non-phosphorylated STAT1, STAT2, and STAT3 were analyzed by western blotting (A). Treatment with 1000 units IFN-α was used as a positive control. Expression of IFIT3 in DV-infected A549 cells with knockdown of either STAT2 (B), STAT1 (C) or STAT3 (D) was determined by western blotting (B, C, and D) or quantitative RT/PCR (B). Both shRNA and siRNA were used as the approaches for STAT1/STAT2 and STAT3, respectively, as described in Materials and Methods. Knockdown with shGFP or si-Ctl was used as a negative control. Data show representative results and analysis pooled from at least 3 independent experiments. The analysis was performed by ANOVA as described in Materials and Methods. **P

    Techniques Used: Infection, Western Blot, Positive Control, Expressing, Quantitative RT-PCR, shRNA, CTL Assay, Negative Control

    16) Product Images from "Interdependent and independent roles of type I interferons and IL-6 in innate immune, neuroinflammatory and sickness behaviour responses to systemic poly I:C"

    Article Title: Interdependent and independent roles of type I interferons and IL-6 in innate immune, neuroinflammatory and sickness behaviour responses to systemic poly I:C

    Journal: Brain, Behavior, and Immunity

    doi: 10.1016/j.bbi.2015.04.009

    Expression of total and phosphorylated STAT1 in WT and IFNAR1 −/− mice. Western blot of basal STAT1 in WT and IFNAR1 −/− hippocampal homogenates (a). Western blot analysis of STAT1 and phosphorylated STAT1 in WT and IFNAR1 −/− mice post-poly I:C challenge at 1 and 3 h (b). Quantitative histogram of basal STAT1 in WT and IFNAR1 −/− mice and levels of STAT1 at 1 and 3 h post-poly I:C (c). Significant differences by Students t test are denoted by ∗∗ p = 0.0015. Expression of stat1 mRNA (d). Data are expressed as mean ± SEM and analysed by two-way ANOVA with treatment and strain as factors; n = 5 for WT + saline, n = 4 for WT + pI:C 1 h, n = 5 for WT + pI:C 3 h and n = 5 for IFNAR1 −/− + saline, n = 4 for IFNAR1 −/− + pI:C 1 h, n = 5 for IFNAR1 −/− + pI:C 3 h. Significant main effect of treatment by Bonferroni post-hoc is denoted by ∗∗∗ p
    Figure Legend Snippet: Expression of total and phosphorylated STAT1 in WT and IFNAR1 −/− mice. Western blot of basal STAT1 in WT and IFNAR1 −/− hippocampal homogenates (a). Western blot analysis of STAT1 and phosphorylated STAT1 in WT and IFNAR1 −/− mice post-poly I:C challenge at 1 and 3 h (b). Quantitative histogram of basal STAT1 in WT and IFNAR1 −/− mice and levels of STAT1 at 1 and 3 h post-poly I:C (c). Significant differences by Students t test are denoted by ∗∗ p = 0.0015. Expression of stat1 mRNA (d). Data are expressed as mean ± SEM and analysed by two-way ANOVA with treatment and strain as factors; n = 5 for WT + saline, n = 4 for WT + pI:C 1 h, n = 5 for WT + pI:C 3 h and n = 5 for IFNAR1 −/− + saline, n = 4 for IFNAR1 −/− + pI:C 1 h, n = 5 for IFNAR1 −/− + pI:C 3 h. Significant main effect of treatment by Bonferroni post-hoc is denoted by ∗∗∗ p

    Techniques Used: Expressing, Mouse Assay, Western Blot

    17) Product Images from "MiR-103 protects from recurrent spontaneous abortion via inhibiting STAT1 mediated M1 macrophage polarization"

    Article Title: MiR-103 protects from recurrent spontaneous abortion via inhibiting STAT1 mediated M1 macrophage polarization

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.46144

    miR-103 inhibits the STAT1/IRF1 signal pathway. RAW264.7 and PM cells were transfected with miR-103 mimics/NC or miR-103 inhibitor/INC, after 24h, the cells were stimulated with or without LPS/IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 mimics or NC by qRT-PCR. ( C-D ) The protein levels of STAT1, p-STAT1 and IRF1 were measured in RAW264.7 and PM cells transfected with miR-103 mimics or NC by western blot. ( E-F ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by qRT-PCR. ( G-H ) STAT1, p-STAT1 and IRF1 protein levels were measured in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by western blot. ( I-J ) Expression of STAT1 and IRF1 were detected in RAW264.7 cells transfected with miR-103 mimics/NC or miR-103 inhibitor/INC by immunofluorescence. DAPI was used to stain the cell nucleus (Scale bar, 50 µm, 200×). Values were listed as the mean± SEM. * P
    Figure Legend Snippet: miR-103 inhibits the STAT1/IRF1 signal pathway. RAW264.7 and PM cells were transfected with miR-103 mimics/NC or miR-103 inhibitor/INC, after 24h, the cells were stimulated with or without LPS/IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 mimics or NC by qRT-PCR. ( C-D ) The protein levels of STAT1, p-STAT1 and IRF1 were measured in RAW264.7 and PM cells transfected with miR-103 mimics or NC by western blot. ( E-F ) STAT1 and IRF1 mRNA expression were detected in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by qRT-PCR. ( G-H ) STAT1, p-STAT1 and IRF1 protein levels were measured in RAW264.7 and PM cells transfected with miR-103 inhibitor or INC by western blot. ( I-J ) Expression of STAT1 and IRF1 were detected in RAW264.7 cells transfected with miR-103 mimics/NC or miR-103 inhibitor/INC by immunofluorescence. DAPI was used to stain the cell nucleus (Scale bar, 50 µm, 200×). Values were listed as the mean± SEM. * P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

    miR-103 suppresses embryo resorption rate and M1 macrophages polarization in vivo . Mice were inspected every morning for vaginal plugs. The day when a plug became visible was designated as Day 0.5 of pregnancy. NP mice and RSA mice were administrated 10 nmol agomiR NC or agomiR-103 on Day 0.5, 3.5, 6.5, 9.5 via tail vein, and execute mice on Day 11.5 of pregnancy. ( A ) Treatment regime of agomiR-103 or agomiR NC and timeline for the measurement of parameters. ( B ) Relative expression of miR-103 was measured by qRT-PCR in the decidua of pregnant mice (n= 10). ( C-D ) Embryo resorption rate of three group mice, arrows indicate the embryo resorption (n= 10). ( E-F ) The mRNA level of STAT1, IRF1 and protein level of p-STAT1, STAT1 and IRF1 were detected in the decidua of pregnant mice (n= 10). ( G ) Dot plot represents labeling of F4/80 + CD80 + (M1) and F4/80 + MHCII + (M1) cell by flow cytometry in the decidua of pregnant mice (n= 10). ( H ) Relative expression of CCL2 , CXCL9 , CXCL10 , iNOS , IL6 , TNF-α was analysed by qRT-PCR in the decidua of pregnant mice (n= 10). Values were listed as the mean± SEM. * P
    Figure Legend Snippet: miR-103 suppresses embryo resorption rate and M1 macrophages polarization in vivo . Mice were inspected every morning for vaginal plugs. The day when a plug became visible was designated as Day 0.5 of pregnancy. NP mice and RSA mice were administrated 10 nmol agomiR NC or agomiR-103 on Day 0.5, 3.5, 6.5, 9.5 via tail vein, and execute mice on Day 11.5 of pregnancy. ( A ) Treatment regime of agomiR-103 or agomiR NC and timeline for the measurement of parameters. ( B ) Relative expression of miR-103 was measured by qRT-PCR in the decidua of pregnant mice (n= 10). ( C-D ) Embryo resorption rate of three group mice, arrows indicate the embryo resorption (n= 10). ( E-F ) The mRNA level of STAT1, IRF1 and protein level of p-STAT1, STAT1 and IRF1 were detected in the decidua of pregnant mice (n= 10). ( G ) Dot plot represents labeling of F4/80 + CD80 + (M1) and F4/80 + MHCII + (M1) cell by flow cytometry in the decidua of pregnant mice (n= 10). ( H ) Relative expression of CCL2 , CXCL9 , CXCL10 , iNOS , IL6 , TNF-α was analysed by qRT-PCR in the decidua of pregnant mice (n= 10). Values were listed as the mean± SEM. * P

    Techniques Used: In Vivo, Mouse Assay, Expressing, Quantitative RT-PCR, Labeling, Flow Cytometry

    Overexpression of STAT1 can reverse the inhibitory effect of miR-103 on M1 polarization. RAW264.7 cells were co-transfected with miR-103 mimics, STAT1 plasmid or NC, vector for 24 h, followed by treating with LPS plus IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA levels were assessed by qRT-PCR. ( C ) p-STAT1, STAT1 and IRF1 protein levels were assessed by western blot. ( D ) mRNA expression level of M1 macrophages makers CXCL10 , IL6 , IL12b , iNOS were detected by qRT-PCR. Values were listed as the mean± SEM. * P
    Figure Legend Snippet: Overexpression of STAT1 can reverse the inhibitory effect of miR-103 on M1 polarization. RAW264.7 cells were co-transfected with miR-103 mimics, STAT1 plasmid or NC, vector for 24 h, followed by treating with LPS plus IFNγ for 24 h. ( A-B ) STAT1 and IRF1 mRNA levels were assessed by qRT-PCR. ( C ) p-STAT1, STAT1 and IRF1 protein levels were assessed by western blot. ( D ) mRNA expression level of M1 macrophages makers CXCL10 , IL6 , IL12b , iNOS were detected by qRT-PCR. Values were listed as the mean± SEM. * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing

    miR-103 was down-regulated in RSA patients and negatively correlated with STAT1 expression. ( A ) Decreased expression of 30 miRNAs ( P
    Figure Legend Snippet: miR-103 was down-regulated in RSA patients and negatively correlated with STAT1 expression. ( A ) Decreased expression of 30 miRNAs ( P

    Techniques Used: Expressing

    STAT1 is a direct target of miR-103. ( A ) Schematic representation of miR-103 putative binding sequence in the 3'UTR of STAT1 , luciferase activities of wild-type (WT) and mutant (MUT) constructs. ( B-C ) The luciferase activity was determined by co-transfecting the vectors ( STAT1 3'UTR-WT or STAT1 3'UTR-MUT) combined with NC, miR-103 mimics into 293T cells. Values were listed as the mean± SEM. *** P
    Figure Legend Snippet: STAT1 is a direct target of miR-103. ( A ) Schematic representation of miR-103 putative binding sequence in the 3'UTR of STAT1 , luciferase activities of wild-type (WT) and mutant (MUT) constructs. ( B-C ) The luciferase activity was determined by co-transfecting the vectors ( STAT1 3'UTR-WT or STAT1 3'UTR-MUT) combined with NC, miR-103 mimics into 293T cells. Values were listed as the mean± SEM. *** P

    Techniques Used: Binding Assay, Sequencing, Luciferase, Mutagenesis, Construct, Activity Assay

    M1 macrophage and STAT1 were excessive in RSA patients. ( A-B ) The dot plot represents labeling of CD14 + CD86 + and CD14 + TNFα + (M1) cells by flow cytometry in decidua of NP subjects (n= 30) and RSA patients (n= 30). ( C ) qRT-PCR analysis of STAT1 expression in the decidua of NP subjects (n= 30) and RSA patients (n= 30). ( D ) STAT1 and p-STAT1 protein levels were measured in decidua of NP subjects (n= 10) and RSA patients (n= 10) by western blot. ( E ) Representative IHC staining images of STAT1 in the decidua of NP and RSA patients (Scale bar, 50 µm, 200×). ( F ) Correlation between p-STAT1 and the proportion of CD14 + CD86 + in decidua of NP subjects (n= 10) and RSA patients (n= 10). ( G ) Correlation between p-STAT1 and the proportion of CD14 + TNF-α + in decidua of NP subjects (n= 10) and RSA patients (n= 10). Values were listed as the mean± SEM. **** P
    Figure Legend Snippet: M1 macrophage and STAT1 were excessive in RSA patients. ( A-B ) The dot plot represents labeling of CD14 + CD86 + and CD14 + TNFα + (M1) cells by flow cytometry in decidua of NP subjects (n= 30) and RSA patients (n= 30). ( C ) qRT-PCR analysis of STAT1 expression in the decidua of NP subjects (n= 30) and RSA patients (n= 30). ( D ) STAT1 and p-STAT1 protein levels were measured in decidua of NP subjects (n= 10) and RSA patients (n= 10) by western blot. ( E ) Representative IHC staining images of STAT1 in the decidua of NP and RSA patients (Scale bar, 50 µm, 200×). ( F ) Correlation between p-STAT1 and the proportion of CD14 + CD86 + in decidua of NP subjects (n= 10) and RSA patients (n= 10). ( G ) Correlation between p-STAT1 and the proportion of CD14 + TNF-α + in decidua of NP subjects (n= 10) and RSA patients (n= 10). Values were listed as the mean± SEM. **** P

    Techniques Used: Labeling, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Staining

    18) Product Images from "PCBP2 Enhances the Antiviral Activity of IFN-? against HCV by Stabilizing the mRNA of STAT1 and STAT2"

    Article Title: PCBP2 Enhances the Antiviral Activity of IFN-? against HCV by Stabilizing the mRNA of STAT1 and STAT2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025419

    The STAT1 and STAT2 mRNA is stabilized in cells overexpressing PCBP2. (A) qRT-PCR was carried out to measure mRNA level of STAT1 and STAT2 after 6 hours of IFN-α treatment. R1b cells were untransfected or transfected with pcDEF-PCBP2. Each bar represents the average of triplicate data points with the standard deviation represented as the error bar . ** P
    Figure Legend Snippet: The STAT1 and STAT2 mRNA is stabilized in cells overexpressing PCBP2. (A) qRT-PCR was carried out to measure mRNA level of STAT1 and STAT2 after 6 hours of IFN-α treatment. R1b cells were untransfected or transfected with pcDEF-PCBP2. Each bar represents the average of triplicate data points with the standard deviation represented as the error bar . ** P

    Techniques Used: Quantitative RT-PCR, Transfection, Standard Deviation

    PCBP2 enhances the effect of IFN-α through binding STAT1 and STAT2 mRNA and up-regulates the expression of the two signal molecules in IFN-α pathway. RIP assay and targeted qRT-PCR confirmed the enrichment of different IFN-α signal pathway factor mRNAs in the PCBP2-RNA complex. (A) Detection of the PCBP2-RNA complex precipitated with streptavidin beads using anti-PCBP2 or anti-GFP antibody and streptavidin by Western blotting. (B) Targeted qRT-PCR was carried out on RNA isolated from RNP complexes precipitated in RIP assay. Analysis of mRNAs isolated from a GFP RIP reaction was carried out in parallel as nonspecific binding. α-globin was used as positive control. The enrichment of each mRNA was compared to the GFP control. Each bar represents the average of triplicate data points with standard deviation represented as the error bar . (C) PCBP2 up-regulated the expression of STAT1 and STAT2 after the treatment of IFN-α while other factors remained intact. The R1b cells were transfected with pcDEF vector or pcDEF-PCBP2. Forty-eight hours after transfection, the cells were treated with 100 IU/mL IFN-α and paired with untrasfected cells. Next, 6 hours later, the cell lysates were assessed by Western blotting.
    Figure Legend Snippet: PCBP2 enhances the effect of IFN-α through binding STAT1 and STAT2 mRNA and up-regulates the expression of the two signal molecules in IFN-α pathway. RIP assay and targeted qRT-PCR confirmed the enrichment of different IFN-α signal pathway factor mRNAs in the PCBP2-RNA complex. (A) Detection of the PCBP2-RNA complex precipitated with streptavidin beads using anti-PCBP2 or anti-GFP antibody and streptavidin by Western blotting. (B) Targeted qRT-PCR was carried out on RNA isolated from RNP complexes precipitated in RIP assay. Analysis of mRNAs isolated from a GFP RIP reaction was carried out in parallel as nonspecific binding. α-globin was used as positive control. The enrichment of each mRNA was compared to the GFP control. Each bar represents the average of triplicate data points with standard deviation represented as the error bar . (C) PCBP2 up-regulated the expression of STAT1 and STAT2 after the treatment of IFN-α while other factors remained intact. The R1b cells were transfected with pcDEF vector or pcDEF-PCBP2. Forty-eight hours after transfection, the cells were treated with 100 IU/mL IFN-α and paired with untrasfected cells. Next, 6 hours later, the cell lysates were assessed by Western blotting.

    Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Positive Control, Standard Deviation, Transfection, Plasmid Preparation

    Model of PCBP2-modulating effect of IFN-α against HCV. PCBP2 enhances the antiviral activity of IFN-α through stabilizing the mRNA of STAT1 and STAT2. This accordingly, increases the protein level of the two molecules. The protein level increase also enhances the effect of IFN-α against HCV.
    Figure Legend Snippet: Model of PCBP2-modulating effect of IFN-α against HCV. PCBP2 enhances the antiviral activity of IFN-α through stabilizing the mRNA of STAT1 and STAT2. This accordingly, increases the protein level of the two molecules. The protein level increase also enhances the effect of IFN-α against HCV.

    Techniques Used: Activity Assay

    The C-rich tracts in the 3′UTR of mRNA of STAT1 and STAT2 are PCBP2 binding sites. (A) Fragments of the STAT1 and STAT2 3′-UTR used in synthesis of sense RNA probe in vitro and sequences position in the 3′-UTR. (B) RNA pull-down assay using the four probes of STAT1 and STAT2. Cytoplasmic extracts from 100 IU/mL IFN-α treated R1b cells were incubated with biotin-labeled RNA probes. The mixture was pulled down by streptavidin beads. The resulting complexes were separated by SDS-PAGE and detected by Western blotting. A pGEM-3zf vector sequence was utilized as a nonspecific control (NSC), and the α-globin 3′-UTR was utilized as a positive control. The bottom panel depicts the assay results with the RNAs set of fragments shown in A . (C) 2-fold, 5-fold and 10-fold of excess unlabeled cold probes were added to compete with the labeled probes for competition analysis. The results indicate that 5-fold cold competitors of S13U2 and S23U1 probe exhibited strong competition. (D) The nucleotide sequences of S13U2 and S23U1. The CU-rich patches are underlined.
    Figure Legend Snippet: The C-rich tracts in the 3′UTR of mRNA of STAT1 and STAT2 are PCBP2 binding sites. (A) Fragments of the STAT1 and STAT2 3′-UTR used in synthesis of sense RNA probe in vitro and sequences position in the 3′-UTR. (B) RNA pull-down assay using the four probes of STAT1 and STAT2. Cytoplasmic extracts from 100 IU/mL IFN-α treated R1b cells were incubated with biotin-labeled RNA probes. The mixture was pulled down by streptavidin beads. The resulting complexes were separated by SDS-PAGE and detected by Western blotting. A pGEM-3zf vector sequence was utilized as a nonspecific control (NSC), and the α-globin 3′-UTR was utilized as a positive control. The bottom panel depicts the assay results with the RNAs set of fragments shown in A . (C) 2-fold, 5-fold and 10-fold of excess unlabeled cold probes were added to compete with the labeled probes for competition analysis. The results indicate that 5-fold cold competitors of S13U2 and S23U1 probe exhibited strong competition. (D) The nucleotide sequences of S13U2 and S23U1. The CU-rich patches are underlined.

    Techniques Used: Binding Assay, In Vitro, Pull Down Assay, Incubation, Labeling, SDS Page, Western Blot, Plasmid Preparation, Sequencing, Positive Control

    19) Product Images from "Nipah Virus V and W Proteins Have a Common STAT1-Binding Domain yet Inhibit STAT1 Activation from the Cytoplasmic and Nuclear Compartments, Respectively"

    Article Title: Nipah Virus V and W Proteins Have a Common STAT1-Binding Domain yet Inhibit STAT1 Activation from the Cytoplasmic and Nuclear Compartments, Respectively

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.11.5633-5641.2004

    Nipah virus V, W, and P all inhibit IFN-induced phosphorylation of STAT1. (A) HeLa cells were transfected with a STAT1 plasmid and an HA-tagged V, W, or P plasmid. STAT1 phosphorylation was induced by treatment with 100 ng of IFN-α/ml for 30 min. Proteins were stained with antibodies against the HA epitope (left panels) or pY701-STAT1 (center panels). In cells expressing V or W, there was no evidence of phosphorylated STAT1 in the nucleus (white arrows). Among cells expressing P, some had activated STAT1 in the nucleus (blue arrows), while others did not (yellow arrows). Merged images are shown in the panels on the right. (B) 293T cells were transfected with the indicated plasmids. One day later, STAT1 phosphorylation was induced by treatment with 1,000 IU of IFN-β/ml for 30 min. Cells were lysed, and levels of pY701-STAT1 (upper panel) and bulk STAT1 (lower panel) were determined by Western blot analysis.
    Figure Legend Snippet: Nipah virus V, W, and P all inhibit IFN-induced phosphorylation of STAT1. (A) HeLa cells were transfected with a STAT1 plasmid and an HA-tagged V, W, or P plasmid. STAT1 phosphorylation was induced by treatment with 100 ng of IFN-α/ml for 30 min. Proteins were stained with antibodies against the HA epitope (left panels) or pY701-STAT1 (center panels). In cells expressing V or W, there was no evidence of phosphorylated STAT1 in the nucleus (white arrows). Among cells expressing P, some had activated STAT1 in the nucleus (blue arrows), while others did not (yellow arrows). Merged images are shown in the panels on the right. (B) 293T cells were transfected with the indicated plasmids. One day later, STAT1 phosphorylation was induced by treatment with 1,000 IU of IFN-β/ml for 30 min. Cells were lysed, and levels of pY701-STAT1 (upper panel) and bulk STAT1 (lower panel) were determined by Western blot analysis.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Expressing, Western Blot

    Amino acids 50 to 150 constitute the smallest domain that is able to interact with STAT1. 293T cells were transfected with a STAT1 expression construct and the indicated Nipah virus construct. Protein complexes were coimmunoprecipitated with an antibody against the HA or V5 epitope, and the interacting protein was analyzed by SDS-PAGE and immunoblotting with an anti-STAT1 antibody. The position of STAT1 is indicated.
    Figure Legend Snippet: Amino acids 50 to 150 constitute the smallest domain that is able to interact with STAT1. 293T cells were transfected with a STAT1 expression construct and the indicated Nipah virus construct. Protein complexes were coimmunoprecipitated with an antibody against the HA or V5 epitope, and the interacting protein was analyzed by SDS-PAGE and immunoblotting with an anti-STAT1 antibody. The position of STAT1 is indicated.

    Techniques Used: Transfection, Expressing, Construct, SDS Page

    Nipah virus W is localized to the nucleus and causes redistribution of STAT1 to the nucleus, whereas V and P localize to the cytoplasm and retain STAT1 in the cytoplasm. HeLa cells were transfected with the STAT1 expression plasmid and HA-tagged V, W, or P. Cells were fixed and permeabilized 1 day later. Proteins were stained with antibodies against the HA epitope (left panels) or STAT1 (center panels), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (right panels).
    Figure Legend Snippet: Nipah virus W is localized to the nucleus and causes redistribution of STAT1 to the nucleus, whereas V and P localize to the cytoplasm and retain STAT1 in the cytoplasm. HeLa cells were transfected with the STAT1 expression plasmid and HA-tagged V, W, or P. Cells were fixed and permeabilized 1 day later. Proteins were stained with antibodies against the HA epitope (left panels) or STAT1 (center panels), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (right panels).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Staining

    20) Product Images from "Rapid Inflammation in Mice Lacking Both SOCS1 and SOCS3 in Hematopoietic Cells"

    Article Title: Rapid Inflammation in Mice Lacking Both SOCS1 and SOCS3 in Hematopoietic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0162111

    Interleukin-6 signalling in mice lacking SOCS proteins. Western blot analysis of macrophages prepared from mice 14d after tamoxifen or vehicle treatment. The cells were stimulated with IL-6 and lysates were prepared at the times indicated. Proteins were separated by polyacrylamide gel electrophoresis, transferred to membranes and probed with antibodies to the molecules indicated at the right. Replicate filters were prepared from the same lysates and probed with pSTAT1 and pSTAT3 and subsequently with STAT1 and STAT3 and SOCS3. Images were scanned and cropped to prepare the Figure; for uncropped images see S6 Fig .
    Figure Legend Snippet: Interleukin-6 signalling in mice lacking SOCS proteins. Western blot analysis of macrophages prepared from mice 14d after tamoxifen or vehicle treatment. The cells were stimulated with IL-6 and lysates were prepared at the times indicated. Proteins were separated by polyacrylamide gel electrophoresis, transferred to membranes and probed with antibodies to the molecules indicated at the right. Replicate filters were prepared from the same lysates and probed with pSTAT1 and pSTAT3 and subsequently with STAT1 and STAT3 and SOCS3. Images were scanned and cropped to prepare the Figure; for uncropped images see S6 Fig .

    Techniques Used: Mouse Assay, Western Blot, Polyacrylamide Gel Electrophoresis

    21) Product Images from "Neuropilin-1 modulates interferon-γ-stimulated signaling in brain microvascular endothelial cells"

    Article Title: Neuropilin-1 modulates interferon-γ-stimulated signaling in brain microvascular endothelial cells

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.190702

    Knockdown of NRP1 inhibited IFNγ-stimulated expression of CXCL10 and activation of STAT1. . (A) HBMVECs were transfected with control siRNA (Csi) or siRNA against NRP1 (Nsi) and then exposed to recombinant IFNγ (10 ng ml −1 ) for 24 h. Expression of CXCL10 was quantified by using qPCR and expressed as a relative fold of that in the control group, which was normalized to 1. n =5 for each group. (B) HBMVECs were transfected with control siRNA (Csi) or NRP1 siRNA (Nsi) for 24 h and then stimulated with IFNγ (10 ng ml −1 ) for 15 min. Total protein lysates were collected and subjected to western blotting with the indicated antibodies. Results are representative of five independent experiments. The bar graph shows the activation of STAT1 expressed as ratios of phosphorylated (p)STAT1 to total STAT1 and compared with that of the control group, which was normalized to 1. (C) The activity of Rac1 was examined in the lysates from the HBMVECs that had been transfected with control siRNA (Csi) or NRP1 siRNA (Nsi) for 24 h and stimulated with IFNγ (10 ng ml −1 . (D) HBMVECs were pretreated with Rac1 inhibitor NSC 23766 (NSC) for 1 h and then stimulated with IFNγ (10 ng ml −1 ) for 24 h. Total RNA was extracted and subjected to qPCR analysis with primers against CXCL10 and β-actin genes. The expressed levels of CXCL10 are reported as a relative fold of the control group, which was normalized to 1. n =4 for each group. (E) HBMVECs were infected with a lentivirus expressing Rac1 61L or LacZ as control for 24 h and then transfected with control siRNA (Csi) or NRP1 siRNA (Nsi) for another 24 h. Total protein lysates were collected after stimulation with IFNγ (10 ng ml −1 ) for 15 min and subjected to western blotting for the indicated proteins. The bar graph shows the activation of STAT1 expressed as the ratios of pSTAT1 to total STAT1, and values were compared with those of the control group, which were normalized to 1. n =4 for each group. * P
    Figure Legend Snippet: Knockdown of NRP1 inhibited IFNγ-stimulated expression of CXCL10 and activation of STAT1. . (A) HBMVECs were transfected with control siRNA (Csi) or siRNA against NRP1 (Nsi) and then exposed to recombinant IFNγ (10 ng ml −1 ) for 24 h. Expression of CXCL10 was quantified by using qPCR and expressed as a relative fold of that in the control group, which was normalized to 1. n =5 for each group. (B) HBMVECs were transfected with control siRNA (Csi) or NRP1 siRNA (Nsi) for 24 h and then stimulated with IFNγ (10 ng ml −1 ) for 15 min. Total protein lysates were collected and subjected to western blotting with the indicated antibodies. Results are representative of five independent experiments. The bar graph shows the activation of STAT1 expressed as ratios of phosphorylated (p)STAT1 to total STAT1 and compared with that of the control group, which was normalized to 1. (C) The activity of Rac1 was examined in the lysates from the HBMVECs that had been transfected with control siRNA (Csi) or NRP1 siRNA (Nsi) for 24 h and stimulated with IFNγ (10 ng ml −1 . (D) HBMVECs were pretreated with Rac1 inhibitor NSC 23766 (NSC) for 1 h and then stimulated with IFNγ (10 ng ml −1 ) for 24 h. Total RNA was extracted and subjected to qPCR analysis with primers against CXCL10 and β-actin genes. The expressed levels of CXCL10 are reported as a relative fold of the control group, which was normalized to 1. n =4 for each group. (E) HBMVECs were infected with a lentivirus expressing Rac1 61L or LacZ as control for 24 h and then transfected with control siRNA (Csi) or NRP1 siRNA (Nsi) for another 24 h. Total protein lysates were collected after stimulation with IFNγ (10 ng ml −1 ) for 15 min and subjected to western blotting for the indicated proteins. The bar graph shows the activation of STAT1 expressed as the ratios of pSTAT1 to total STAT1, and values were compared with those of the control group, which were normalized to 1. n =4 for each group. * P

    Techniques Used: Expressing, Activation Assay, Transfection, Recombinant, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Infection

    22) Product Images from "Context-enriched interactome powered by proteomics helps the identification of novel regulators of macrophage activation"

    Article Title: Context-enriched interactome powered by proteomics helps the identification of novel regulators of macrophage activation

    Journal: eLife

    doi: 10.7554/eLife.37059

    ( A ) Spearman correlation values between the gene expression and protein abundance for the top 2826 candidates for M(IFNγ) and the top 4109 candidates for M(IL-4), based on maximizing the sum of sensitivity and specificity. ( B ) Putative feedback regulation of GBP1 on the JAK2-STAT1-CCL2 signaling pathway. ( C ) Abundance profiles of GBP family of genes found in the proteomics measurements.
    Figure Legend Snippet: ( A ) Spearman correlation values between the gene expression and protein abundance for the top 2826 candidates for M(IFNγ) and the top 4109 candidates for M(IL-4), based on maximizing the sum of sensitivity and specificity. ( B ) Putative feedback regulation of GBP1 on the JAK2-STAT1-CCL2 signaling pathway. ( C ) Abundance profiles of GBP family of genes found in the proteomics measurements.

    Techniques Used: Expressing

    23) Product Images from "Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway"

    Article Title: Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway

    Journal: Journal of cancer science & therapy

    doi: 10.4172/1948-5956.1000310

    Effect of STAT1 inhibition on cell motility with IL-27 and apricoxib combination. (A) After treatment of A549 cells with IL-27 (50 ng/mL) at 60-70% confluence, a scratch was made in a monolayer of cells and the closure of the wound gap was observed under
    Figure Legend Snippet: Effect of STAT1 inhibition on cell motility with IL-27 and apricoxib combination. (A) After treatment of A549 cells with IL-27 (50 ng/mL) at 60-70% confluence, a scratch was made in a monolayer of cells and the closure of the wound gap was observed under

    Techniques Used: Inhibition

    Effect of apricoxib on IL-27 mediated STAT activation. (A) A549 cells were treated with apricoxib (0.016-2 μM) for 4 hours prior to IL-27 exposure IL-27 (50 ng/mL) for 15 minutes, and the tyrosine phosphorylated forms of STAT1 and STAT3 (P-STAT1;
    Figure Legend Snippet: Effect of apricoxib on IL-27 mediated STAT activation. (A) A549 cells were treated with apricoxib (0.016-2 μM) for 4 hours prior to IL-27 exposure IL-27 (50 ng/mL) for 15 minutes, and the tyrosine phosphorylated forms of STAT1 and STAT3 (P-STAT1;

    Techniques Used: Activation Assay

    Role of STAT1 in the enhancing effect of apricoxib and IL-27 on the inhibition of EMT. A549 cells were treated with a STAT1 siRNA (40 nM) or a non-targeting control siRNA (40 nM) for 6 hours prior to treatment with IL-27 (50 ng/mL) for 15 minutes and/or
    Figure Legend Snippet: Role of STAT1 in the enhancing effect of apricoxib and IL-27 on the inhibition of EMT. A549 cells were treated with a STAT1 siRNA (40 nM) or a non-targeting control siRNA (40 nM) for 6 hours prior to treatment with IL-27 (50 ng/mL) for 15 minutes and/or

    Techniques Used: Inhibition

    24) Product Images from "Human Cytomegalovirus Modulates Monocyte-Mediated Innate Immune Responses during Short-Term Experimental Latency In Vitro"

    Article Title: Human Cytomegalovirus Modulates Monocyte-Mediated Innate Immune Responses during Short-Term Experimental Latency In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.00934-14

    Latent HCMV restricts interferon signaling at the level of STAT1 phosphorylation. (A) CD14 + monocytes that had been mock infected or TB40/E infected were treated at day 3 postinfection with 1,000 U/ml of IFN-β for 30 min and then harvested for
    Figure Legend Snippet: Latent HCMV restricts interferon signaling at the level of STAT1 phosphorylation. (A) CD14 + monocytes that had been mock infected or TB40/E infected were treated at day 3 postinfection with 1,000 U/ml of IFN-β for 30 min and then harvested for

    Techniques Used: Infection

    25) Product Images from "Hepatocyte-Specific Deletion of Mouse Lamin A/C Leads to Male-Selective Steatohepatitis"

    Article Title: Hepatocyte-Specific Deletion of Mouse Lamin A/C Leads to Male-Selective Steatohepatitis

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2017.06.005

    Absence of lamin A/C in hepatocytes interferes with GH signaling in male hepatocytes. The schematic summarizes our findings. Lmna deficiency is responsible for the following: (1) alters nuclear shape; (2) inhibits growth hormone receptor–mediated Jak2, Stat5, and Erk phosphorylation; (3) decreases the expression of Stat5-dependent male-specific genes; (4) increases expression of the Stat5-regulated fatty acid translocator CD36, (5) up-regulates Cidea transcription, and (6) increases Stat1 messenger RNA and protein expression and activation, resulting in the induction of interferon-regulated genes. These alterations lead to hepatocyte storage of excess fatty acids with consequent induction of hepatic inflammation and fibrosis upon feeding with a HFD.
    Figure Legend Snippet: Absence of lamin A/C in hepatocytes interferes with GH signaling in male hepatocytes. The schematic summarizes our findings. Lmna deficiency is responsible for the following: (1) alters nuclear shape; (2) inhibits growth hormone receptor–mediated Jak2, Stat5, and Erk phosphorylation; (3) decreases the expression of Stat5-dependent male-specific genes; (4) increases expression of the Stat5-regulated fatty acid translocator CD36, (5) up-regulates Cidea transcription, and (6) increases Stat1 messenger RNA and protein expression and activation, resulting in the induction of interferon-regulated genes. These alterations lead to hepatocyte storage of excess fatty acids with consequent induction of hepatic inflammation and fibrosis upon feeding with a HFD.

    Techniques Used: Expressing, Activation Assay

    Hepatic Lmna deficiency causes up-regulation of Stat1 expression and phosphorylation in male liver. ( A ) Stat1 qPCR analysis of messenger RNA from WT/Het/KO male livers of mice (age, 20–25 wk) fed ND or HFD (* P
    Figure Legend Snippet: Hepatic Lmna deficiency causes up-regulation of Stat1 expression and phosphorylation in male liver. ( A ) Stat1 qPCR analysis of messenger RNA from WT/Het/KO male livers of mice (age, 20–25 wk) fed ND or HFD (* P

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

    Hepatocyte-specific Lmna -deficiency promotes liver inflammation. ( A ) Three Lmna Het and 3 Lmna KO livers from male mice fed a normal diet were compared using Affymetrix microarrays (same as those described in Figure 7 ). Het and KO livers were isolated from 26-week-old and 39-week-old mice, respectively. Transcripts that were significantly different and carried the gene ontology term immune were grouped together in a heat map (red, high expression; blue, low expression as compared with the mean values for the WT livers). Black arrows denote interferon-regulated genes that are up-regulated in KO livers. The red arrow indicates up-regulated Stat1 expression in KO livers. ( B ) qPCR analysis of messenger RNA from livers of WT and KO male mice (23–28 weeks old, 5 livers/genotype) fed ND or HFD. Expression of the indicated inflammation-associated genes is shown (* P
    Figure Legend Snippet: Hepatocyte-specific Lmna -deficiency promotes liver inflammation. ( A ) Three Lmna Het and 3 Lmna KO livers from male mice fed a normal diet were compared using Affymetrix microarrays (same as those described in Figure 7 ). Het and KO livers were isolated from 26-week-old and 39-week-old mice, respectively. Transcripts that were significantly different and carried the gene ontology term immune were grouped together in a heat map (red, high expression; blue, low expression as compared with the mean values for the WT livers). Black arrows denote interferon-regulated genes that are up-regulated in KO livers. The red arrow indicates up-regulated Stat1 expression in KO livers. ( B ) qPCR analysis of messenger RNA from livers of WT and KO male mice (23–28 weeks old, 5 livers/genotype) fed ND or HFD. Expression of the indicated inflammation-associated genes is shown (* P

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

    26) Product Images from "Different Associations of CD45 Isoforms with STAT3, PKC and ERK Regulate IL-6-Induced Proliferation in Myeloma"

    Article Title: Different Associations of CD45 Isoforms with STAT3, PKC and ERK Regulate IL-6-Induced Proliferation in Myeloma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119780

    IL-6-induced S.TAT3 and STAT1 nuclear translocation is required for the integrity of lipid rafts. (A) CD45 + U266 cells were transfected with untagged (mock) or STAT1-EGFP, STAT3-EGFP expression plasmids, and either treated or untreated with IL-6 at different time points. Immunoblotting was performed as above. (B) Subcellular distribution of STATs-EGFP fusion proteins. Nuclear translocation of both STAT3-EGFP and STAT1-EGFP was evaluated by live cell imaging. Cells were pre-incubated with or without 10 mM MCD for 30 minutes and then stimulated with 10 ng/ml of IL-6 and images were generated at different time points. P-REP was used as a mock vector, which expresses EGFP. Data shown are representative of three experiments.
    Figure Legend Snippet: IL-6-induced S.TAT3 and STAT1 nuclear translocation is required for the integrity of lipid rafts. (A) CD45 + U266 cells were transfected with untagged (mock) or STAT1-EGFP, STAT3-EGFP expression plasmids, and either treated or untreated with IL-6 at different time points. Immunoblotting was performed as above. (B) Subcellular distribution of STATs-EGFP fusion proteins. Nuclear translocation of both STAT3-EGFP and STAT1-EGFP was evaluated by live cell imaging. Cells were pre-incubated with or without 10 mM MCD for 30 minutes and then stimulated with 10 ng/ml of IL-6 and images were generated at different time points. P-REP was used as a mock vector, which expresses EGFP. Data shown are representative of three experiments.

    Techniques Used: Translocation Assay, Transfection, Expressing, Live Cell Imaging, Incubation, Generated, Plasmid Preparation

    IL-6-induced STAT3 and STAT1 phosphorylation is required for the integrity of lipid rafts. (A) CD45 + U266 cells were grown in IL-6 free medium for 12 hours (IL-6 starvation). Cells were incubated with or without 10 ng/ml of IL-6 for 5 minutes. The cell lysates were subjected to sucrose density gradient centrifugation, and endogenous proteins indicated beside figures from each sucrose fraction were analyzed by immunoblotting. CD71 was detected as a nonraft marker. Lipid raft fractions were confirmed using CTX dot plots for each fraction. (B) CD45 + U266 cells were treated or untreated with MCD (10 mM) for 30 minutes at 37°C. Cells were then stimulated with IL-6 at different time points. Whole-cell lysates were subjected to SDS/PAGE and separate plots with antibodies are shown. The representative blots of three independent experiments are shown.
    Figure Legend Snippet: IL-6-induced STAT3 and STAT1 phosphorylation is required for the integrity of lipid rafts. (A) CD45 + U266 cells were grown in IL-6 free medium for 12 hours (IL-6 starvation). Cells were incubated with or without 10 ng/ml of IL-6 for 5 minutes. The cell lysates were subjected to sucrose density gradient centrifugation, and endogenous proteins indicated beside figures from each sucrose fraction were analyzed by immunoblotting. CD71 was detected as a nonraft marker. Lipid raft fractions were confirmed using CTX dot plots for each fraction. (B) CD45 + U266 cells were treated or untreated with MCD (10 mM) for 30 minutes at 37°C. Cells were then stimulated with IL-6 at different time points. Whole-cell lysates were subjected to SDS/PAGE and separate plots with antibodies are shown. The representative blots of three independent experiments are shown.

    Techniques Used: Incubation, Gradient Centrifugation, Marker, SDS Page

    27) Product Images from "4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression"

    Article Title: 4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-018-2172-2

    Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis
    Figure Legend Snippet: Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis

    Techniques Used: Activation Assay, Concentration Assay, Western Blot

    28) Product Images from "AG490 suppresses EPO-mediated activation of JAK2-STAT but enhances blood flow recovery in rats with critical limb ischemia"

    Article Title: AG490 suppresses EPO-mediated activation of JAK2-STAT but enhances blood flow recovery in rats with critical limb ischemia

    Journal: Journal of Inflammation (London, England)

    doi: 10.1186/s12950-016-0126-3

    EPO regulates the expression and phosphorylation levels of JAK/STATs in quadriceps after critical limb ischemia. a Twenty-four hours after CLI, total protein was extracted from quadriceps and Western blots were performed with antibodies against JAK2, phospho-JAK2, STAT5, phospho-STAT5, STAT1, phospho-STAT1, STAT3, and phospho-STAT3. b and c The expression levels of total and phosphorylated JAK2. d and e The expression levels of total and phosphorylated STAT1. f and g The expression levels of total and phosphorylated STAT3. h and i The expression levels of total and phosphorylated STAT5. Statistical analysis used one-way ANOVA followed by Bonferroni multiple comparison post hoc test ( n = 8 for each group). Symbols (*, †, ‡,) indicate significance at p value less than 0.05. EPO, Erythropoietin; JAK, Janus kinase; STAT, Signal Transducer and Activator of Transcription
    Figure Legend Snippet: EPO regulates the expression and phosphorylation levels of JAK/STATs in quadriceps after critical limb ischemia. a Twenty-four hours after CLI, total protein was extracted from quadriceps and Western blots were performed with antibodies against JAK2, phospho-JAK2, STAT5, phospho-STAT5, STAT1, phospho-STAT1, STAT3, and phospho-STAT3. b and c The expression levels of total and phosphorylated JAK2. d and e The expression levels of total and phosphorylated STAT1. f and g The expression levels of total and phosphorylated STAT3. h and i The expression levels of total and phosphorylated STAT5. Statistical analysis used one-way ANOVA followed by Bonferroni multiple comparison post hoc test ( n = 8 for each group). Symbols (*, †, ‡,) indicate significance at p value less than 0.05. EPO, Erythropoietin; JAK, Janus kinase; STAT, Signal Transducer and Activator of Transcription

    Techniques Used: Expressing, Western Blot

    29) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

    Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113318

    CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
    Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

    Techniques Used: Amplification, Isolation, Genome Wide, Expressing

    IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

    Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

    STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Isolation, Quantitative RT-PCR

    30) Product Images from "High Glucose and Interferon Gamma Synergistically Stimulate MMP-1 Expression in U937 Macrophages by Increasing Transcription Factor STAT1 Activity"

    Article Title: High Glucose and Interferon Gamma Synergistically Stimulate MMP-1 Expression in U937 Macrophages by Increasing Transcription Factor STAT1 Activity

    Journal: Atherosclerosis

    doi: 10.1016/j.atherosclerosis.2008.05.043

    Enhancement of IFNγ-induced nuclear STAT1 DNA-binding activity by high glucose. U937 macrophages cultured in normal or high glucose-containing medium were treated with 100 units/ml of IFNγ for 2, 4, 8, or 24 h. After the treatment, nuclear protein was isolated from cells and subject to STAT1 (A) and STAT3 (C) DNA-binding activity assay, and STAT1 electrophoretic mobility shift assay (B) as described in Methods. The data presented are the representative of two independent experiments with the similar results.
    Figure Legend Snippet: Enhancement of IFNγ-induced nuclear STAT1 DNA-binding activity by high glucose. U937 macrophages cultured in normal or high glucose-containing medium were treated with 100 units/ml of IFNγ for 2, 4, 8, or 24 h. After the treatment, nuclear protein was isolated from cells and subject to STAT1 (A) and STAT3 (C) DNA-binding activity assay, and STAT1 electrophoretic mobility shift assay (B) as described in Methods. The data presented are the representative of two independent experiments with the similar results.

    Techniques Used: Binding Assay, Activity Assay, Cell Culture, Isolation, Electrophoretic Mobility Shift Assay

    31) Product Images from "Designing Cell-Targeted Therapeutic Proteins Reveals the Interplay between Domain Connectivity and Cell Binding"

    Article Title: Designing Cell-Targeted Therapeutic Proteins Reveals the Interplay between Domain Connectivity and Cell Binding

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2014.10.007

    Simulation across a range of receptor binding affinities predicts a region of maximal targeting effect. We performed ODE simulations for a wide range of physiologically plausible k on IFNAR1 and k on IFNAR2 values to predict Stat1 phosphorylation
    Figure Legend Snippet: Simulation across a range of receptor binding affinities predicts a region of maximal targeting effect. We performed ODE simulations for a wide range of physiologically plausible k on IFNAR1 and k on IFNAR2 values to predict Stat1 phosphorylation

    Techniques Used: Binding Assay

    The SNAP-tagged proteins are biologically active. ( A ) Stat1 phosphorylation in K562 CD20+ cells induced by SNAP-IFN fusion proteins after 30 min. Both IFN α (WT)-SNAP and IFN α (R144A mutant)-SNAP induced robust Stat1 phosphorylation
    Figure Legend Snippet: The SNAP-tagged proteins are biologically active. ( A ) Stat1 phosphorylation in K562 CD20+ cells induced by SNAP-IFN fusion proteins after 30 min. Both IFN α (WT)-SNAP and IFN α (R144A mutant)-SNAP induced robust Stat1 phosphorylation

    Techniques Used: Mutagenesis

    32) Product Images from "Hepatitis C Virus Nonstructural Protein 5A Modulates the Toll-Like Receptor-MyD88-Dependent Signaling Pathway in Macrophage Cell Lines ▿"

    Article Title: Hepatitis C Virus Nonstructural Protein 5A Modulates the Toll-Like Receptor-MyD88-Dependent Signaling Pathway in Macrophage Cell Lines ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00649-07

    ISDR in NS5A participates in the inhibition of the MyD88-dependent signaling pathway. (A) Structures of NS5A mutants lacking amino acid residues 240 to 280, in which the ISDR/MyD88-interacting region is located (Δ240-280), and lacking amino acid residues 280 to 300 (Δ280-300) (left). Immunoblot analyses of cells expressing wild-type or mutant NS5A (right) are shown. (B) Cells expressing wild-type or mutant NS5A were stimulated with the indicated amounts of mCpG, R-837, LPS, or PGN, and the production of IL-6 in the culture supernatants was determined by ELISA 24 h after stimulation. Data are shown as the means ± SD. (C) Phosphorylation of STAT1 or PKR in response to treatment with murine IFN-α or infection with VSV. The cell lines were stimulated with two doses of murine IFN-α (2 × 10 3 and 2 × 10 2 units/ml) or VSV (2 × 10 7 and 2 × 10 6 PFU/ml). After 24 h of stimulation, cell extracts were immunoblotted (IB) with specific antibodies. Phosphorylated STAT1 and PKR and the total amounts of STAT1 and β-actin were determined. The asterisk indicates nonspecific bands.
    Figure Legend Snippet: ISDR in NS5A participates in the inhibition of the MyD88-dependent signaling pathway. (A) Structures of NS5A mutants lacking amino acid residues 240 to 280, in which the ISDR/MyD88-interacting region is located (Δ240-280), and lacking amino acid residues 280 to 300 (Δ280-300) (left). Immunoblot analyses of cells expressing wild-type or mutant NS5A (right) are shown. (B) Cells expressing wild-type or mutant NS5A were stimulated with the indicated amounts of mCpG, R-837, LPS, or PGN, and the production of IL-6 in the culture supernatants was determined by ELISA 24 h after stimulation. Data are shown as the means ± SD. (C) Phosphorylation of STAT1 or PKR in response to treatment with murine IFN-α or infection with VSV. The cell lines were stimulated with two doses of murine IFN-α (2 × 10 3 and 2 × 10 2 units/ml) or VSV (2 × 10 7 and 2 × 10 6 PFU/ml). After 24 h of stimulation, cell extracts were immunoblotted (IB) with specific antibodies. Phosphorylated STAT1 and PKR and the total amounts of STAT1 and β-actin were determined. The asterisk indicates nonspecific bands.

    Techniques Used: Inhibition, Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection

    33) Product Images from "IFN gamma regulates proliferation and neuronal differentiation by STAT1 in adult SVZ niche"

    Article Title: IFN gamma regulates proliferation and neuronal differentiation by STAT1 in adult SVZ niche

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2015.00270

    IFNγ modulates proliferation and neuronal differentiation in adult SVZ and causes a final neurogenesis impairment in the olfactory bulb(OB) in vivo . (A) Phosphorylation of STAT1 in the ipsilateral (Ip) and contralateral (ctr) SVZ niche of adult mice 30 min after they received an intracerebroventricular (ICV) injection of IFNγ (1 μl of 50 ng/ml) ( n = 3). (B) Pictures illustrating the decrease of Ki67 in dorsal SVZ after in vivo gain of function of IFNγ ( n = 4). (C) Histogram representing the total number of positive cells in the dorsal SVZ. BrdU+ cells and Ki67+ cells decreased in IFNγ-gain animals. (D) Pictures of dorsal SVZ showing substantial decrease in Nestin+ cells and mild differences in DCX+ cells of IFNγ-gain animals. (E) Quantification summarizes the robust decrease in Nestin+ progenitor cells and the mild reduction in neuroblast populations (DCX+ or PSA-NCAM+) in the SVZ niche of IFNγ-treated animals ( n = 6). (F) Histograms summarizing cellular fate determination (expressed as percentage) in animals injected with BrdU at time 0 and with a pump implanted for 4d. Neuronal fate determination (BrdU+/DCX+) was significantly increased and glial production (BrdU+/Olig2+) was unaltered by IFNγ ( n = 5). (G) Pictures of the OB showing the presence of BrdU+, NeuN+ and BrdU+/NeuN+ labeled cells in IFNγ-gain mice. The number of newborn cells (BrdU) and neurons (BrdU/NeuN) were reduced in the animals in which IFNγ activity was induced. (H) Histograms summarizing the quantification of total number of newborn cells and neurons per section in the OB of controls and IFNγ-gain animals ( n = 5). The number of newborn cells (BrdU+) and neurons (BrdU+/NeuN+) was reduced in the presence of IFNγ. Scale bar: (B, D) and (G) 100 μm. Data are represented as mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.01.
    Figure Legend Snippet: IFNγ modulates proliferation and neuronal differentiation in adult SVZ and causes a final neurogenesis impairment in the olfactory bulb(OB) in vivo . (A) Phosphorylation of STAT1 in the ipsilateral (Ip) and contralateral (ctr) SVZ niche of adult mice 30 min after they received an intracerebroventricular (ICV) injection of IFNγ (1 μl of 50 ng/ml) ( n = 3). (B) Pictures illustrating the decrease of Ki67 in dorsal SVZ after in vivo gain of function of IFNγ ( n = 4). (C) Histogram representing the total number of positive cells in the dorsal SVZ. BrdU+ cells and Ki67+ cells decreased in IFNγ-gain animals. (D) Pictures of dorsal SVZ showing substantial decrease in Nestin+ cells and mild differences in DCX+ cells of IFNγ-gain animals. (E) Quantification summarizes the robust decrease in Nestin+ progenitor cells and the mild reduction in neuroblast populations (DCX+ or PSA-NCAM+) in the SVZ niche of IFNγ-treated animals ( n = 6). (F) Histograms summarizing cellular fate determination (expressed as percentage) in animals injected with BrdU at time 0 and with a pump implanted for 4d. Neuronal fate determination (BrdU+/DCX+) was significantly increased and glial production (BrdU+/Olig2+) was unaltered by IFNγ ( n = 5). (G) Pictures of the OB showing the presence of BrdU+, NeuN+ and BrdU+/NeuN+ labeled cells in IFNγ-gain mice. The number of newborn cells (BrdU) and neurons (BrdU/NeuN) were reduced in the animals in which IFNγ activity was induced. (H) Histograms summarizing the quantification of total number of newborn cells and neurons per section in the OB of controls and IFNγ-gain animals ( n = 5). The number of newborn cells (BrdU+) and neurons (BrdU+/NeuN+) was reduced in the presence of IFNγ. Scale bar: (B, D) and (G) 100 μm. Data are represented as mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.01.

    Techniques Used: In Vivo, Mouse Assay, Injection, Labeling, Activity Assay

    IFNγ promotes migration from SVZ explants via STAT1. (A) Brightfield pictures showing that IFNγ increases the migration area of progenitors exiting SVZ postnatal explants and the absence of STAT1 reverted IFNγ effects ( n = 4). (B) Histograms showing the ratio of migration area of proximal (P) vs. distal(D) regions of the explants, in WT and KO samples, in the presence of IFNγ ( n = 5). Scale bars = 100 μm. Data are represented as mean ± SEM. * p ≤ 0.05.
    Figure Legend Snippet: IFNγ promotes migration from SVZ explants via STAT1. (A) Brightfield pictures showing that IFNγ increases the migration area of progenitors exiting SVZ postnatal explants and the absence of STAT1 reverted IFNγ effects ( n = 4). (B) Histograms showing the ratio of migration area of proximal (P) vs. distal(D) regions of the explants, in WT and KO samples, in the presence of IFNγ ( n = 5). Scale bars = 100 μm. Data are represented as mean ± SEM. * p ≤ 0.05.

    Techniques Used: Migration

    IFNγ regulates proliferation and differentiation of NSCs from SVZ through STAT1. (A) Brightfield pictures showing how the number of secondary neurospheres was dramatically reduced by IFNγ and that STAT1 prevents cytokine actions. (B) Histogram showing how IFNγ impedes neurosphere formation through STAT1 ( n = 5). (C) Histogram demostrating the presence of neural cells after 7-day NSCs culture differentiation. The presence of IFNγ did not affect viability (PI incorporation); whereas the number of KI67+ and original Nestin+ cells decreased, and the number of committed neuroblasts (TUBB3+ cells) increased, without affecting oligodendroglial differentiation (NG2+ cells) or the number of GFAP+ cells ( n = 5). (D) Photographs illustrating the increased neurogenesis induced by IFNγ through STAT1. IFNγ induced a higher neuroblast (red) differentiation, which reverted in the absence of STAT1. Hoechst (blue) stained all nuclei ( n = 5). (E) Histogram demonstrating the presence of neuroblasts (TUBB3+ cells) originated from neural 7-day NSC culture differentiation. IFNγ improves neuronal differentiation in WT cultures but not in STAT1 deficient cells ( n = 5) in which a slightly reduction in neuroblasts was detected. Scale bar: (A) , 100 μm and (D) , 40 μm. Data are represented as mean ± SEM. * p
    Figure Legend Snippet: IFNγ regulates proliferation and differentiation of NSCs from SVZ through STAT1. (A) Brightfield pictures showing how the number of secondary neurospheres was dramatically reduced by IFNγ and that STAT1 prevents cytokine actions. (B) Histogram showing how IFNγ impedes neurosphere formation through STAT1 ( n = 5). (C) Histogram demostrating the presence of neural cells after 7-day NSCs culture differentiation. The presence of IFNγ did not affect viability (PI incorporation); whereas the number of KI67+ and original Nestin+ cells decreased, and the number of committed neuroblasts (TUBB3+ cells) increased, without affecting oligodendroglial differentiation (NG2+ cells) or the number of GFAP+ cells ( n = 5). (D) Photographs illustrating the increased neurogenesis induced by IFNγ through STAT1. IFNγ induced a higher neuroblast (red) differentiation, which reverted in the absence of STAT1. Hoechst (blue) stained all nuclei ( n = 5). (E) Histogram demonstrating the presence of neuroblasts (TUBB3+ cells) originated from neural 7-day NSC culture differentiation. IFNγ improves neuronal differentiation in WT cultures but not in STAT1 deficient cells ( n = 5) in which a slightly reduction in neuroblasts was detected. Scale bar: (A) , 100 μm and (D) , 40 μm. Data are represented as mean ± SEM. * p

    Techniques Used: Staining

    STAT1 mediates IFNγ activities in vivo . (A) Quantification summarizes how, in the absence of STAT1, proliferation (BrdU) and the expression of neuronal markers such a DCX and olig2 were unaltered. (B) Histogram representing a similar proportion of newborn determined neurons (BrdU+/DCX+) and glial cells in the SVZ of STAT1-KO animals that received a single BrdU 4 days before sacrifice, ( n = 6). (C) Pictures of the SVZ sections illustrating that STAT1 deficiency impeded IFNγ functions in the in vivo model. The numbers of proliferative cells (BrdU, green) and neuroblasts (red) were unchanged in KO samples in the IFNγ-treated animals. (D) Quantification summarizes how IFNγ effects on proliferation (BrdU incorporation) and neuroblast populations (DCX+ cells) were abolished in the absence of STAT1 mediator ( n = 6). (E) Histogram representing the impossibility of IFNγ inducing neuronal differentiation (BrdU+/DCX+ cells) in STAT1-KO animals that received a single BrdU administration 4 days before sacrifice. ( n = 6). Scale bar: 50 μm. Data are represented as mean ± SEM. * p
    Figure Legend Snippet: STAT1 mediates IFNγ activities in vivo . (A) Quantification summarizes how, in the absence of STAT1, proliferation (BrdU) and the expression of neuronal markers such a DCX and olig2 were unaltered. (B) Histogram representing a similar proportion of newborn determined neurons (BrdU+/DCX+) and glial cells in the SVZ of STAT1-KO animals that received a single BrdU 4 days before sacrifice, ( n = 6). (C) Pictures of the SVZ sections illustrating that STAT1 deficiency impeded IFNγ functions in the in vivo model. The numbers of proliferative cells (BrdU, green) and neuroblasts (red) were unchanged in KO samples in the IFNγ-treated animals. (D) Quantification summarizes how IFNγ effects on proliferation (BrdU incorporation) and neuroblast populations (DCX+ cells) were abolished in the absence of STAT1 mediator ( n = 6). (E) Histogram representing the impossibility of IFNγ inducing neuronal differentiation (BrdU+/DCX+ cells) in STAT1-KO animals that received a single BrdU administration 4 days before sacrifice. ( n = 6). Scale bar: 50 μm. Data are represented as mean ± SEM. * p

    Techniques Used: In Vivo, Expressing, BrdU Incorporation Assay

    IFNγ increases tyrosine STAT1 phosphorylation in Nestin+ SVZ progenitors. (A) Phosphorylation of STAT1 at Tyr701 and JAK2 were induced 15 min after IFNγ (50 ng/mL) addition in primary SVZ cultures. Stimulation of pSTAT3 Tyr701 , pSTAT3 Ser727 , and JAK1 were not observed ( n = 6 per time point). Total STAT1 and STAT3 were unchanged and actin was the loading control. (B) Double-immunofluorescences staining showed that STAT1 Tyr701 immunoreactivity was absent in the control culture. After IFNγ stimulation STAT1 Tyr701 (green) was increased exclusively in Nestin+ progenitors (red; n = 4), TO-PRO (blue) labeled all nuclei. (C) The presence of the AG490 STAT1 inhibitor reduced STAT1 phosphorylation ( n = 4 per time point). Actin was the loading control. (D) Histogram represents immunoreactivity signal intensity of STAT1 Tyr701 per Nestin+ cell after IFNγ stimulation in the presence or absence of AG490 inhibitor. After IFNγ stimulation, phosphorylation of STAT1S Tyr701 was increased in Nestin+ progenitors and the presence of the JAK2 phosphorylation inhibitor AG490 impeded STAT1 phosphorylation ( n = 4). Scale bar: (B) 30 μm. Data are represented as mean ± SEM. ** p ≤ 0.01.
    Figure Legend Snippet: IFNγ increases tyrosine STAT1 phosphorylation in Nestin+ SVZ progenitors. (A) Phosphorylation of STAT1 at Tyr701 and JAK2 were induced 15 min after IFNγ (50 ng/mL) addition in primary SVZ cultures. Stimulation of pSTAT3 Tyr701 , pSTAT3 Ser727 , and JAK1 were not observed ( n = 6 per time point). Total STAT1 and STAT3 were unchanged and actin was the loading control. (B) Double-immunofluorescences staining showed that STAT1 Tyr701 immunoreactivity was absent in the control culture. After IFNγ stimulation STAT1 Tyr701 (green) was increased exclusively in Nestin+ progenitors (red; n = 4), TO-PRO (blue) labeled all nuclei. (C) The presence of the AG490 STAT1 inhibitor reduced STAT1 phosphorylation ( n = 4 per time point). Actin was the loading control. (D) Histogram represents immunoreactivity signal intensity of STAT1 Tyr701 per Nestin+ cell after IFNγ stimulation in the presence or absence of AG490 inhibitor. After IFNγ stimulation, phosphorylation of STAT1S Tyr701 was increased in Nestin+ progenitors and the presence of the JAK2 phosphorylation inhibitor AG490 impeded STAT1 phosphorylation ( n = 4). Scale bar: (B) 30 μm. Data are represented as mean ± SEM. ** p ≤ 0.01.

    Techniques Used: Staining, Labeling

    34) Product Images from "Differential Requirement for TANK-binding Kinase-1 in Type I Interferon Responses to Toll-like Receptor Activation and Viral Infection"

    Article Title: Differential Requirement for TANK-binding Kinase-1 in Type I Interferon Responses to Toll-like Receptor Activation and Viral Infection

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20040528

    TBK1-deficient BMMs have defective type I IFN responses to polyI:C. TBK1 +/+ TNFR1 +/− and TBK1 −/− TNFR1 −/− BMMs were stimulated with 1 μg/ml polyI:C for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control. (B) Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IFNα5, IRF7, IL-15, and Mx1.
    Figure Legend Snippet: TBK1-deficient BMMs have defective type I IFN responses to polyI:C. TBK1 +/+ TNFR1 +/− and TBK1 −/− TNFR1 −/− BMMs were stimulated with 1 μg/ml polyI:C for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control. (B) Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IFNα5, IRF7, IL-15, and Mx1.

    Techniques Used: Polymerase Chain Reaction, Expressing

    TBK1-deficient MEF cells have impaired IFN responses to SeV. Wild-type and TBK1 −/− MEF cells were infected with SeV for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control. Total cell extracts were probed for phospho-STAT1 and total STAT1. (B) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IRF7, and IL-15. (C) EMSA was performed by incubating nuclear extracts were with an NF-κB-specific oligonucleotide. (D) Nuclear fractions were probed for p65 and USF2. (E) Total RNA was extracted and analyzed by Q-PCR for expression of ICAM1 and IκBα.
    Figure Legend Snippet: TBK1-deficient MEF cells have impaired IFN responses to SeV. Wild-type and TBK1 −/− MEF cells were infected with SeV for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control. Total cell extracts were probed for phospho-STAT1 and total STAT1. (B) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IRF7, and IL-15. (C) EMSA was performed by incubating nuclear extracts were with an NF-κB-specific oligonucleotide. (D) Nuclear fractions were probed for p65 and USF2. (E) Total RNA was extracted and analyzed by Q-PCR for expression of ICAM1 and IκBα.

    Techniques Used: Infection, Polymerase Chain Reaction, Expressing

    TBK1-deficient BMMs have normal IFN responses to SeV. TBK1 +/+ TNFR1 +/− and TBK1 −/− TNFR1 −/− BMMs were infected with SeV for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control. (B) Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IFNα5, IRF7, IL-15, and Mx1.
    Figure Legend Snippet: TBK1-deficient BMMs have normal IFN responses to SeV. TBK1 +/+ TNFR1 +/− and TBK1 −/− TNFR1 −/− BMMs were infected with SeV for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control. (B) Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IFNα5, IRF7, IL-15, and Mx1.

    Techniques Used: Infection, Polymerase Chain Reaction, Expressing

    TBK1 is required for LPS-mediated activation of type I IFN responses. TBK1 +/+ TNFR1 +/− and TBK1 −/− TNFR1 −/− BMMs were stimulated with 10 ng/ml LPS for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control for nuclear proteins. (B) Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IFNα5, IRF7, IL-15, and Mx1.
    Figure Legend Snippet: TBK1 is required for LPS-mediated activation of type I IFN responses. TBK1 +/+ TNFR1 +/− and TBK1 −/− TNFR1 −/− BMMs were stimulated with 10 ng/ml LPS for the indicated time points. (A) Nuclear fractions were probed for IRF3 and USF2 as a loading control for nuclear proteins. (B) Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IP-10, IFNα5, IRF7, IL-15, and Mx1.

    Techniques Used: Activation Assay, Polymerase Chain Reaction, Expressing

    IKK- i is differentially expressed in BMMs and MEFs, and can rescue a TBK1 deficiency during SeV infection. (A) Total RNA extracted from BMMs, wild-type MEFs, and TBK1 −/− MEFs were probed by Northern blot for expression of TBK1 and IKK- i . 28 and 18 sRNA bands are shown as loading controls. (B) Wild-type MEFs, TBK1 −/− MEFs, and TBK1 −/− MEFs reconstituted with wild-type TBK1 or IKK- i , or their kinase-inactive (KA) mutants were infected with SeV for the time points indicated. Nuclear fractions were probed for IRF3, p65, and USF2 as a loading control. Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Cells were infected with SeV as in B and total RNA was extracted and analyzed by Q-PCR for expression of IFNβ.
    Figure Legend Snippet: IKK- i is differentially expressed in BMMs and MEFs, and can rescue a TBK1 deficiency during SeV infection. (A) Total RNA extracted from BMMs, wild-type MEFs, and TBK1 −/− MEFs were probed by Northern blot for expression of TBK1 and IKK- i . 28 and 18 sRNA bands are shown as loading controls. (B) Wild-type MEFs, TBK1 −/− MEFs, and TBK1 −/− MEFs reconstituted with wild-type TBK1 or IKK- i , or their kinase-inactive (KA) mutants were infected with SeV for the time points indicated. Nuclear fractions were probed for IRF3, p65, and USF2 as a loading control. Total cell extracts were probed for phospho-STAT1 and total STAT1. (C) Cells were infected with SeV as in B and total RNA was extracted and analyzed by Q-PCR for expression of IFNβ.

    Techniques Used: Infection, Northern Blot, Expressing, Polymerase Chain Reaction

    35) Product Images from "Bortezomib Pre-Treatment Prolongs Interferon-alpha Induced STAT1 Phosphorylation in Melanoma Cells"

    Article Title: Bortezomib Pre-Treatment Prolongs Interferon-alpha Induced STAT1 Phosphorylation in Melanoma Cells

    Journal: Cancer immunology, immunotherapy : CII

    doi: 10.1007/s00262-009-0710-y

    Pre-treatment of melanoma cells with bortezomib leads to enhanced phosphorylation of STAT1 in response to IFN-α
    Figure Legend Snippet: Pre-treatment of melanoma cells with bortezomib leads to enhanced phosphorylation of STAT1 in response to IFN-α

    Techniques Used:

    36) Product Images from "Hepatitis C Virus Infection Impairs IRF-7 Translocation and Alpha Interferon Synthesis in Immortalized Human Hepatocytes ▿"

    Article Title: Hepatitis C Virus Infection Impairs IRF-7 Translocation and Alpha Interferon Synthesis in Immortalized Human Hepatocytes ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00900-10

    STAT1 downregulation enhances HCV genome replication. (A) IHH were transfected with STAT1 siRNA or control (scrambled) siRNA. Cells were lysed after 5 days posttransfection, and STAT1 protein was analyzed by Western blotting using specific antibody. (B) STAT1-downregulated cells were infected with HCV genotype 1a or genotype 2a at an MOI of 0.1. Total cellular RNA was extracted 72 h postinfection, and HCV RNA was analyzed by quantitative RT-PCR. The results shown are means from three independent experiments.
    Figure Legend Snippet: STAT1 downregulation enhances HCV genome replication. (A) IHH were transfected with STAT1 siRNA or control (scrambled) siRNA. Cells were lysed after 5 days posttransfection, and STAT1 protein was analyzed by Western blotting using specific antibody. (B) STAT1-downregulated cells were infected with HCV genotype 1a or genotype 2a at an MOI of 0.1. Total cellular RNA was extracted 72 h postinfection, and HCV RNA was analyzed by quantitative RT-PCR. The results shown are means from three independent experiments.

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

    HCV infection induces STAT1 expression in hepatocytes. (A) IHH were infected with HCV genotype 1a or genotype 2a and incubated for 10 days. Western blot analysis was performed for STAT1 or phospho-STAT1 protein expression with specific antibodies. The blot was reprobed with an antibody to tubulin for comparison of protein loads. Mock- and HCV-infected IHH were also treated with IFN-α (400 units) for 1 h as a positive control to evaluate the phopsho-STAT1 expression status. (B) IHH were either mock treated (a), transfected with poly(I-C) as a positive control (b), or infected with HCV genotype 1a (c to f). Mock-treated and poly(I-C)-treated cells were fixed and stained with STAT1 antibody (red) after 24 h. HCV-infected IHH were fixed at day 10 postinfection and stained for STAT1 (red) and HCV NS5A (green). Nuclei were visualized by staining with 4′,6-diamidino-2-phenylindole (blue). Merged images from panels c to e are shown in panel f. Arrowheads indicate the selected uninfected cells, and arrows point to HCV-infected cells showing green fluorescence.
    Figure Legend Snippet: HCV infection induces STAT1 expression in hepatocytes. (A) IHH were infected with HCV genotype 1a or genotype 2a and incubated for 10 days. Western blot analysis was performed for STAT1 or phospho-STAT1 protein expression with specific antibodies. The blot was reprobed with an antibody to tubulin for comparison of protein loads. Mock- and HCV-infected IHH were also treated with IFN-α (400 units) for 1 h as a positive control to evaluate the phopsho-STAT1 expression status. (B) IHH were either mock treated (a), transfected with poly(I-C) as a positive control (b), or infected with HCV genotype 1a (c to f). Mock-treated and poly(I-C)-treated cells were fixed and stained with STAT1 antibody (red) after 24 h. HCV-infected IHH were fixed at day 10 postinfection and stained for STAT1 (red) and HCV NS5A (green). Nuclei were visualized by staining with 4′,6-diamidino-2-phenylindole (blue). Merged images from panels c to e are shown in panel f. Arrowheads indicate the selected uninfected cells, and arrows point to HCV-infected cells showing green fluorescence.

    Techniques Used: Infection, Expressing, Incubation, Western Blot, Positive Control, Transfection, Staining, Fluorescence

    37) Product Images from "Knockout of cytochrome P450 1A1 enhances lipopolysaccharide‐induced acute lung injury in mice by targeting NF‐κB activation"

    Article Title: Knockout of cytochrome P450 1A1 enhances lipopolysaccharide‐induced acute lung injury in mice by targeting NF‐κB activation

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12977

    CYP1A1 deficiency promoted NF‐κB activation in LPS‐induced ALI. (A‐C) CYP1A1+/+ and CYP1A1−/− mice were intraperitoneally injected with PBS or LPS (15 mg·kg −1 ) for 1, 2, or 6 h. (A) The phosphorylation and total levels of p65 and IκB‐α in lung homogenates were analyzed by western blot after 1 and 2 h of LPS treatment and quantified by densitometry. (B) The DNA‐binding activity of p65 was determined with a TransAM p65 transcription factor ELISA kit ( n = 5 for each group). (C) The phosphorylation and total levels of STAT1 and AP‐1 (jun and fos) in lung homogenates were examined at the indicated times and quantified by densitometry. Group means were compared by Student's t ‐test. Data shown are means ± SEM. * P
    Figure Legend Snippet: CYP1A1 deficiency promoted NF‐κB activation in LPS‐induced ALI. (A‐C) CYP1A1+/+ and CYP1A1−/− mice were intraperitoneally injected with PBS or LPS (15 mg·kg −1 ) for 1, 2, or 6 h. (A) The phosphorylation and total levels of p65 and IκB‐α in lung homogenates were analyzed by western blot after 1 and 2 h of LPS treatment and quantified by densitometry. (B) The DNA‐binding activity of p65 was determined with a TransAM p65 transcription factor ELISA kit ( n = 5 for each group). (C) The phosphorylation and total levels of STAT1 and AP‐1 (jun and fos) in lung homogenates were examined at the indicated times and quantified by densitometry. Group means were compared by Student's t ‐test. Data shown are means ± SEM. * P

    Techniques Used: Activation Assay, Mouse Assay, Injection, Western Blot, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    38) Product Images from "A guanidine-rich regulatory oligodeoxynucleotide improves type-2 diabetes in obese mice by blocking T-cell differentiation"

    Article Title: A guanidine-rich regulatory oligodeoxynucleotide improves type-2 diabetes in obese mice by blocking T-cell differentiation

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201201272

    Immunosuppression of STAT1/3/4 phosphorylation by ODNR01 selective binding A,B. STAT and phospho-STAT Western blot analysis of anti-CD3/28 mAb-stimulated CD4 + T cells treated with indicated cytokines, with and without 5 µM of ODNR01, ODNA151 or ODN1612. C. Confocal microscopy to detect co-localization of FITC-conjugated ODNs (2.5 µM; green) with different Alex Fluor 555-conjugated anti-STAT rabbit polyclonal antibodies (red) in CD4 + T cells after 24 h of incubation. D. Left panels: Immunoblot analysis of STATs from CD4 + T cells pre-incubated with 5 µM biotinylated-ODNs, lysed, and precipitated with avidin agarose beads for STAT detection. Right panels: Immunoblot analysis of STATs from CD4 + T-cell lysates that were pre-incubated with 1 µM biotinylated ODN and 5 µM unlabelled ODN for 1 h and immunoprecipitated with avidin beads for detection of different STAT. The experiments were repeated three times with similar results.
    Figure Legend Snippet: Immunosuppression of STAT1/3/4 phosphorylation by ODNR01 selective binding A,B. STAT and phospho-STAT Western blot analysis of anti-CD3/28 mAb-stimulated CD4 + T cells treated with indicated cytokines, with and without 5 µM of ODNR01, ODNA151 or ODN1612. C. Confocal microscopy to detect co-localization of FITC-conjugated ODNs (2.5 µM; green) with different Alex Fluor 555-conjugated anti-STAT rabbit polyclonal antibodies (red) in CD4 + T cells after 24 h of incubation. D. Left panels: Immunoblot analysis of STATs from CD4 + T cells pre-incubated with 5 µM biotinylated-ODNs, lysed, and precipitated with avidin agarose beads for STAT detection. Right panels: Immunoblot analysis of STATs from CD4 + T-cell lysates that were pre-incubated with 1 µM biotinylated ODN and 5 µM unlabelled ODN for 1 h and immunoprecipitated with avidin beads for detection of different STAT. The experiments were repeated three times with similar results.

    Techniques Used: Binding Assay, Western Blot, Confocal Microscopy, Incubation, Avidin-Biotin Assay, Immunoprecipitation

    39) Product Images from "Peroxisome Proliferator-activated Receptor ? Negatively Regulates IFN-? Production in Toll-like Receptor (TLR) 3- and TLR4-stimulated Macrophages by Preventing Interferon Regulatory Factor 3 Binding to the IFN-? Promoter *"

    Article Title: Peroxisome Proliferator-activated Receptor ? Negatively Regulates IFN-? Production in Toll-like Receptor (TLR) 3- and TLR4-stimulated Macrophages by Preventing Interferon Regulatory Factor 3 Binding to the IFN-? Promoter *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.149823

    PPAR-γ inhibits TLR3- and TLR4-induced activation of STAT1. A and B , mouse peritoneal macrophages were stimulated with 20 μg/ml of poly(I:C) or 100 ng/ml of LPS in the presence of DMSO or troglitazone. Phosphorylated STAT1 (Tyr-701) and total STAT1 in lysates were detected by immunoblot. Similar results were obtained in three independent experiments. C , mouse peritoneal macrophages were transfected with control small RNA ( Ctrl RNAi ) or PPAR-γ siRNA ( PPAR -γ RNAi ). After 36 h, cells were treated as above, and phosphorylated STAT1 (Tyr-701) and total STAT1 in lysates were detected by immunoblot. Similar results were obtained in three independent experiments. D , RAW264.7 cells in 96-well plates (2.5 × 10 4 /well) were transiently co-transfected with 100 ng of ISRE reporter plasmid, 10 ng of pTK- Renilla plasmid, and 100 ng of PPAR-γ expression plasmid or control plasmid. After 24 h, cells were stimulated with 100 ng/ml of LPS or 20 μg/ml of poly(I:C) for 6 h in the DMSO or troglitazone. Luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± S.D. ( n = 6) of one representative experiment (**, p
    Figure Legend Snippet: PPAR-γ inhibits TLR3- and TLR4-induced activation of STAT1. A and B , mouse peritoneal macrophages were stimulated with 20 μg/ml of poly(I:C) or 100 ng/ml of LPS in the presence of DMSO or troglitazone. Phosphorylated STAT1 (Tyr-701) and total STAT1 in lysates were detected by immunoblot. Similar results were obtained in three independent experiments. C , mouse peritoneal macrophages were transfected with control small RNA ( Ctrl RNAi ) or PPAR-γ siRNA ( PPAR -γ RNAi ). After 36 h, cells were treated as above, and phosphorylated STAT1 (Tyr-701) and total STAT1 in lysates were detected by immunoblot. Similar results were obtained in three independent experiments. D , RAW264.7 cells in 96-well plates (2.5 × 10 4 /well) were transiently co-transfected with 100 ng of ISRE reporter plasmid, 10 ng of pTK- Renilla plasmid, and 100 ng of PPAR-γ expression plasmid or control plasmid. After 24 h, cells were stimulated with 100 ng/ml of LPS or 20 μg/ml of poly(I:C) for 6 h in the DMSO or troglitazone. Luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± S.D. ( n = 6) of one representative experiment (**, p

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

    40) Product Images from "Beauvericin Ameliorates Experimental Colitis by Inhibiting Activated T Cells via Downregulation of the PI3K/Akt Signaling Pathway"

    Article Title: Beauvericin Ameliorates Experimental Colitis by Inhibiting Activated T Cells via Downregulation of the PI3K/Akt Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083013

    Effects of beauvericin on cytokine production, STAT1 phosphorylation, and T-bet expression in T lymphocytes. CD3 + T cells purified from lymph nodes (5×10 5 ) were incubated with different concentrations of beauvericin in the presence of Con A for 24 h. The levels of TNF-α, IFN-γ and IL-2 (A) in supernatants of T cells were determined by ELISA (B, C) T cells were cultured with 2.5-10 µM beauvericin for 3 h, then treated with 25 ng/mL murine IFN-γ for 30 min (detection of pSTAT1, B) or 6 h (detection of T-bet, C). After the incubation, proteins were extracted and assessed by western blotting. The values are shown as mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: Effects of beauvericin on cytokine production, STAT1 phosphorylation, and T-bet expression in T lymphocytes. CD3 + T cells purified from lymph nodes (5×10 5 ) were incubated with different concentrations of beauvericin in the presence of Con A for 24 h. The levels of TNF-α, IFN-γ and IL-2 (A) in supernatants of T cells were determined by ELISA (B, C) T cells were cultured with 2.5-10 µM beauvericin for 3 h, then treated with 25 ng/mL murine IFN-γ for 30 min (detection of pSTAT1, B) or 6 h (detection of T-bet, C). After the incubation, proteins were extracted and assessed by western blotting. The values are shown as mean ± SEM from three independent experiments. * P

    Techniques Used: Expressing, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot

    Related Articles

    Incubation:

    Article Title: Leukotriene B4 Receptor-2 Promotes Invasiveness and Metastasis of Ovarian Cancer Cells through Signal Transducer and Activator of Transcription 3 (STAT3)-dependent Up-regulation of Matrix Metalloproteinase 2 *
    Article Snippet: .. The samples were then subjected to SDS-PAGE, and the separated proteins were transferred electrophoretically to a PVDF membrane for 90 min at 100 V. The membrane was exposed for 1 h to TBS containing 0.05% Tween 20 and 5% dried nonfat milk before incubation overnight at 4 °C with antibodies to STAT3, to phosphorylated STAT3 (Tyr705 or Ser727 ), to phosphorylated STAT1 (Tyr701 ), to STAT1, to 5-lipoxygenase (5-LO), to 12-lipoxygenase (12-LO), to FLAP (5-lipoxygenase-activating protein), or to α-tubulin (loading control), all of which were obtained from Cell Signaling Technology (Danvers, MA) and were used at a dilution of 1:2000 in TBS containing 0.05% Tween 20, with the exception of those to α-tubulin (1:4000 dilution). .. The membrane was then incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibodies before detection of immune complexes with the use of an enhanced chemiluminescence kit (Amersham Biosciences).

    Article Title: Signaling and Immunoresolving Actions of Resolvin D1 in Inflamed Human Visceral Adipose Tissue
    Article Snippet: .. Blots were washed 3 times for 5 min each with 0.1% T-TBS and subsequently incubated overnight at 4°C with primary anti-human antibodies for HO-1, SOCS3, phospho-STAT1 (Tyr701), phospho-STAT3 (Tyr705), phospho-p38 MAPK (Thr180/Tyr 182), STAT1, STAT3 and p38 MAPK, all purchased from Cell Signaling. .. Thereafter, the blots were washed 3 times for 5 min each with 0.1% T-TBS containing 5% (w/v) nonfat dry milk and incubated for 1 h at room temperature with donkey anti-rabbit (Biolegend) or anti-mouse (Cell Signaling) HRP-linked antibody (1:2000) in 0.1% T-TBS.

    SDS Page:

    Article Title: Leukotriene B4 Receptor-2 Promotes Invasiveness and Metastasis of Ovarian Cancer Cells through Signal Transducer and Activator of Transcription 3 (STAT3)-dependent Up-regulation of Matrix Metalloproteinase 2 *
    Article Snippet: .. The samples were then subjected to SDS-PAGE, and the separated proteins were transferred electrophoretically to a PVDF membrane for 90 min at 100 V. The membrane was exposed for 1 h to TBS containing 0.05% Tween 20 and 5% dried nonfat milk before incubation overnight at 4 °C with antibodies to STAT3, to phosphorylated STAT3 (Tyr705 or Ser727 ), to phosphorylated STAT1 (Tyr701 ), to STAT1, to 5-lipoxygenase (5-LO), to 12-lipoxygenase (12-LO), to FLAP (5-lipoxygenase-activating protein), or to α-tubulin (loading control), all of which were obtained from Cell Signaling Technology (Danvers, MA) and were used at a dilution of 1:2000 in TBS containing 0.05% Tween 20, with the exception of those to α-tubulin (1:4000 dilution). .. The membrane was then incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibodies before detection of immune complexes with the use of an enhanced chemiluminescence kit (Amersham Biosciences).

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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)
    Non Phosphorylated Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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