phospho stat1 tyr701  (Cell Signaling Technology Inc)

 
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    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.
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    Structured Review

    Cell Signaling Technology Inc phospho stat1 tyr701
    RvD1 attenuates IL-10-induced <t>STAT1</t> activation in human obese adipose tissue and macrophages (A) Levels of STAT1 <t>Tyr701</t> phosphorylation (pSTAT1 Tyr ) after 30 min of IL-10 stimulation and expression of its target gene CXCL9 in obese adipose tissue explants incubated for 6 h with IL-10 (20 ng/ml). (B) Representative inmunoblots of pSTAT1 Tyr and β-actin assessed by western blot in visceral adipose tissue explants incubated with increasing concentrations of RvD1 (0, 1, 10 and 50 nM) for 30 min followed by the addition of IL-10 (20 ng/ml) for 30 or 120 min . (C) Representative immunoblots analyzing STAT1 activation in THP1 monocytes incubated in the presence of increasing concentrations of IL-10 for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. (D) mRNA expression for STAT1 target genes (CXCL9 and CXCL10) in THP1-derived macrophages incubated in the presence of increasing concentrations of IL-10 for 6 h. (E) Representative immunoblots of STAT1 and pSTAT1 Tyr in THP-1-derived macrophages incubated in the presence of increasing concentrations of RvD1 for 30 min followed by the addition of IL-10 (20 ng/ml) for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. Results are expressed as mean ± SEM of n=3 independent experiments performed in duplicate. P values are given vs vehicle. *, P
    The Stat1 transcription factor is activated in response to a large number of ligands 1 and is essential for responsiveness to IFN α and IFN γ 2 3 Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization nuclear translocation and DNA binding 4 Stat1 protein exists as a pair of isoforms Stat1α 91 kDa and the splice variant Stat1β 84 kDa In most cells both isoforms are activated by IFN α but only Stat1α is activated by IFN γ The inappropriate activation of Stat1 occurs in many tumors 5 In addition to tyrosine phosphorylation Stat1 is also phosphorylated at Ser727 through a p38 mitogen activated protein kinase MAPK dependent pathway in response to IFN α and other cellular stresses 6 Serine phosphorylation may be required for the maximal induction of Stat1 mediated gene activation
    https://www.bioz.com/result/phospho stat1 tyr701/product/Cell Signaling Technology Inc
    Average 98 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    phospho stat1 tyr701 - by Bioz Stars, 2020-11
    98/100 stars

    Images

    1) Product Images from "Signaling and Immunoresolving Actions of Resolvin D1 in Inflamed Human Visceral Adipose Tissue"

    Article Title: Signaling and Immunoresolving Actions of Resolvin D1 in Inflamed Human Visceral Adipose Tissue

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

    doi: 10.4049/jimmunol.1502522

    RvD1 attenuates IL-10-induced STAT1 activation in human obese adipose tissue and macrophages (A) Levels of STAT1 Tyr701 phosphorylation (pSTAT1 Tyr ) after 30 min of IL-10 stimulation and expression of its target gene CXCL9 in obese adipose tissue explants incubated for 6 h with IL-10 (20 ng/ml). (B) Representative inmunoblots of pSTAT1 Tyr and β-actin assessed by western blot in visceral adipose tissue explants incubated with increasing concentrations of RvD1 (0, 1, 10 and 50 nM) for 30 min followed by the addition of IL-10 (20 ng/ml) for 30 or 120 min . (C) Representative immunoblots analyzing STAT1 activation in THP1 monocytes incubated in the presence of increasing concentrations of IL-10 for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. (D) mRNA expression for STAT1 target genes (CXCL9 and CXCL10) in THP1-derived macrophages incubated in the presence of increasing concentrations of IL-10 for 6 h. (E) Representative immunoblots of STAT1 and pSTAT1 Tyr in THP-1-derived macrophages incubated in the presence of increasing concentrations of RvD1 for 30 min followed by the addition of IL-10 (20 ng/ml) for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. Results are expressed as mean ± SEM of n=3 independent experiments performed in duplicate. P values are given vs vehicle. *, P
    Figure Legend Snippet: RvD1 attenuates IL-10-induced STAT1 activation in human obese adipose tissue and macrophages (A) Levels of STAT1 Tyr701 phosphorylation (pSTAT1 Tyr ) after 30 min of IL-10 stimulation and expression of its target gene CXCL9 in obese adipose tissue explants incubated for 6 h with IL-10 (20 ng/ml). (B) Representative inmunoblots of pSTAT1 Tyr and β-actin assessed by western blot in visceral adipose tissue explants incubated with increasing concentrations of RvD1 (0, 1, 10 and 50 nM) for 30 min followed by the addition of IL-10 (20 ng/ml) for 30 or 120 min . (C) Representative immunoblots analyzing STAT1 activation in THP1 monocytes incubated in the presence of increasing concentrations of IL-10 for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. (D) mRNA expression for STAT1 target genes (CXCL9 and CXCL10) in THP1-derived macrophages incubated in the presence of increasing concentrations of IL-10 for 6 h. (E) Representative immunoblots of STAT1 and pSTAT1 Tyr in THP-1-derived macrophages incubated in the presence of increasing concentrations of RvD1 for 30 min followed by the addition of IL-10 (20 ng/ml) for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. Results are expressed as mean ± SEM of n=3 independent experiments performed in duplicate. P values are given vs vehicle. *, P

    Techniques Used: Activation Assay, Expressing, Incubation, Western Blot, Derivative Assay

    2) Product Images from "Tyk2/STAT3 Signaling Mediates β-Amyloid-Induced Neuronal Cell Death: Implications in Alzheimer's Disease"

    Article Title: Tyk2/STAT3 Signaling Mediates β-Amyloid-Induced Neuronal Cell Death: Implications in Alzheimer's Disease

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0519-10.2010

    Tyrosine phosphorylation of STAT3 is elevated in cortex and hippocampus of AD mouse models. A , Cortices, hippocampi, and cerebelli from 12-month-old APP751SL/PS1M146L double-transgenic mouse brains were lysed and subjected to Western blot analysis using p-Tyr705 STAT3 (p-Tyr-STAT3), p-Tyr701 STAT1 (p-Tyr-STAT1), and their corresponding total antibodies. B , Cortices from different ages of APPswe/PS1ΔE9 (designated as APP/PS1) transgenic mice were homogenized, and lysates were subjected to Western blot analysis for p-Tyr-STAT3 and STAT3. C , Paraffin sections of 9-month-old APP/PS1 transgenic mouse brain cortices were stained using p-Tyr-STAT3 antibody, DAPI, and the neuron-specific marker NeuN as indicated. Scale bar, 50 μm. D , Paraffin sections of 12-month-old APP/PS1 transgenic mouse cortices were stained using p-Tyr-STAT3 antibody (brown; DAB) and Aβ antibody (red; AEC). Scale bar, 50 μm. The arrow indicated the p-Tyr STAT3-positive cells, whereas the arrowhead depicted the Aβ deposit.
    Figure Legend Snippet: Tyrosine phosphorylation of STAT3 is elevated in cortex and hippocampus of AD mouse models. A , Cortices, hippocampi, and cerebelli from 12-month-old APP751SL/PS1M146L double-transgenic mouse brains were lysed and subjected to Western blot analysis using p-Tyr705 STAT3 (p-Tyr-STAT3), p-Tyr701 STAT1 (p-Tyr-STAT1), and their corresponding total antibodies. B , Cortices from different ages of APPswe/PS1ΔE9 (designated as APP/PS1) transgenic mice were homogenized, and lysates were subjected to Western blot analysis for p-Tyr-STAT3 and STAT3. C , Paraffin sections of 9-month-old APP/PS1 transgenic mouse brain cortices were stained using p-Tyr-STAT3 antibody, DAPI, and the neuron-specific marker NeuN as indicated. Scale bar, 50 μm. D , Paraffin sections of 12-month-old APP/PS1 transgenic mouse cortices were stained using p-Tyr-STAT3 antibody (brown; DAB) and Aβ antibody (red; AEC). Scale bar, 50 μm. The arrow indicated the p-Tyr STAT3-positive cells, whereas the arrowhead depicted the Aβ deposit.

    Techniques Used: Transgenic Assay, Western Blot, Mouse Assay, Staining, Marker

    3) Product Images from "Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction"

    Article Title: Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20198955

    Model calibration with IFNα‐induced signal transduction in Huh7.5 upon prestimulation and stimulation with IFNα Growth factor‐depleted Huh7.5 cells were prestimulated with 2.8, 28, 1,400 pM IFNα or left untreated and were stimulated with 1,400 pM IFNα 24 h later. IFNα‐induced signaling was measured by time‐resolved quantitative immunoblotting and detected with chemiluminescence using a CCD camera‐based device. Data were normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration. Model calibration with time‐resolved IFNα‐induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data were represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively. Model calibration with time‐resolved IFNα‐induced feedback transcripts upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNα, assessed by qRT‐PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT , and TBP . Experimental data are represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 14) using a combined scaling and error model. Model trajectories are represented by lines. Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9, and USP18 determined 24 h after prestimulation with 0, 2.8, 28, or 1,400 pM IFNα. Calibrator proteins were spiked into 10 μg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD camera‐based device. Averaged values ( N = 4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model. Model calibration of IFNα‐induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8, 28, or 1,400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data are represented by filled circles with errors representing 1σ confidence interval estimated from biological replicates ( N = 4 to N = 22) using a combined scaling and error model. Model trajectories are represented by lines.
    Figure Legend Snippet: Model calibration with IFNα‐induced signal transduction in Huh7.5 upon prestimulation and stimulation with IFNα Growth factor‐depleted Huh7.5 cells were prestimulated with 2.8, 28, 1,400 pM IFNα or left untreated and were stimulated with 1,400 pM IFNα 24 h later. IFNα‐induced signaling was measured by time‐resolved quantitative immunoblotting and detected with chemiluminescence using a CCD camera‐based device. Data were normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration. Model calibration with time‐resolved IFNα‐induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data were represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively. Model calibration with time‐resolved IFNα‐induced feedback transcripts upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNα, assessed by qRT‐PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT , and TBP . Experimental data are represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 14) using a combined scaling and error model. Model trajectories are represented by lines. Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9, and USP18 determined 24 h after prestimulation with 0, 2.8, 28, or 1,400 pM IFNα. Calibrator proteins were spiked into 10 μg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD camera‐based device. Averaged values ( N = 4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model. Model calibration of IFNα‐induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8, 28, or 1,400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data are represented by filled circles with errors representing 1σ confidence interval estimated from biological replicates ( N = 4 to N = 22) using a combined scaling and error model. Model trajectories are represented by lines.

    Techniques Used: Transduction, Quantitative RT-PCR

    4) Product Images from "The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells"

    Article Title: The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0532-y

    UCHL1 inhibition promoted the activation of NF-κB and STAT1 signaling. a After pretreatment with DMSO or LDN57444 (10 μM), murine MSCs were treated with IFN-γ plus TNF-α (10 ng/ml each) for the indicated time. Cells were harvested and NF-κB p65, STAT1, phosphorylation of NF-κB p65 and STAT1 at Tyr701 were analyzed by immunoblotting analysis. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. b After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were treated with or without IFN-γ plus TNF-α (10 ng/ml each) for 6 h. Nucleus protein of cells was harvested and NF-κB p65 was analyzed by immunoblotting analysis. c After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, cells were harvested and IκBα expression was analyzed by immunoblotting analysis. d Control MSCs, vector-MSCs and UCHL1-MSCs were harvested, and IκBα expression was analyzed by immunoblotting analysis. e After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were pretreated with NF-κB inhibitor PDTC (1 μM) or STAT1 inhibitor Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (10 ng/ml each) treatment for 24 h. Cells were harvested and iNOS expression was analyzed by immunoblotting analysis. f , g After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, irradiated untreated, DMSO-pretreated or LDN57444-pretreated murine MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with CFSE-labeled splenocytes for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P
    Figure Legend Snippet: UCHL1 inhibition promoted the activation of NF-κB and STAT1 signaling. a After pretreatment with DMSO or LDN57444 (10 μM), murine MSCs were treated with IFN-γ plus TNF-α (10 ng/ml each) for the indicated time. Cells were harvested and NF-κB p65, STAT1, phosphorylation of NF-κB p65 and STAT1 at Tyr701 were analyzed by immunoblotting analysis. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. b After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were treated with or without IFN-γ plus TNF-α (10 ng/ml each) for 6 h. Nucleus protein of cells was harvested and NF-κB p65 was analyzed by immunoblotting analysis. c After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, cells were harvested and IκBα expression was analyzed by immunoblotting analysis. d Control MSCs, vector-MSCs and UCHL1-MSCs were harvested, and IκBα expression was analyzed by immunoblotting analysis. e After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were pretreated with NF-κB inhibitor PDTC (1 μM) or STAT1 inhibitor Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (10 ng/ml each) treatment for 24 h. Cells were harvested and iNOS expression was analyzed by immunoblotting analysis. f , g After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, irradiated untreated, DMSO-pretreated or LDN57444-pretreated murine MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with CFSE-labeled splenocytes for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P

    Techniques Used: Inhibition, Activation Assay, Software, Expressing, Plasmid Preparation, Irradiation, Co-Culture Assay, Labeling, Flow Cytometry, Cytometry

    UCHL1 inhibition promoted the immunosuppressive capacity and IDO expression of human MSCs. a Human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 12 and 24 h, and UCHL1 expression was examined by immunoblotting analysis. b Human MSCs were pretreated with DMSO or LDN57444 (20 μM) for 24 h, and irradiated untreated, DMSO-pretreated or LDN57444-pretreated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the indicated ratios. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. c After pretreatment with DMSO or LDN57444 (20 μM or 30 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 24 h, and IDO expression was examined by immunoblotting analysis. d After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1, and 1-MT was added to block IDO. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. e After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each), phosphorylation of NF-κB p65 and STAT1 at Tyr701 were examined by immunoblotting analysis at the indicated time points. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. f After pretreatment with DMSO or LDN57444 (20 μM), human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (5 ng/ml each) treatment for 24 h, and IDO expression was examined by immunoblotting assay. g , h After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P
    Figure Legend Snippet: UCHL1 inhibition promoted the immunosuppressive capacity and IDO expression of human MSCs. a Human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 12 and 24 h, and UCHL1 expression was examined by immunoblotting analysis. b Human MSCs were pretreated with DMSO or LDN57444 (20 μM) for 24 h, and irradiated untreated, DMSO-pretreated or LDN57444-pretreated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the indicated ratios. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. c After pretreatment with DMSO or LDN57444 (20 μM or 30 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 24 h, and IDO expression was examined by immunoblotting analysis. d After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1, and 1-MT was added to block IDO. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. e After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each), phosphorylation of NF-κB p65 and STAT1 at Tyr701 were examined by immunoblotting analysis at the indicated time points. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. f After pretreatment with DMSO or LDN57444 (20 μM), human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (5 ng/ml each) treatment for 24 h, and IDO expression was examined by immunoblotting assay. g , h After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P

    Techniques Used: Inhibition, Expressing, Irradiation, Cell Culture, Labeling, Flow Cytometry, Cytometry, Co-Culture Assay, Blocking Assay, Software

    5) Product Images from "The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells"

    Article Title: The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0532-y

    UCHL1 inhibition promoted the activation of NF-κB and STAT1 signaling. a After pretreatment with DMSO or LDN57444 (10 μM), murine MSCs were treated with IFN-γ plus TNF-α (10 ng/ml each) for the indicated time. Cells were harvested and NF-κB p65, STAT1, phosphorylation of NF-κB p65 and STAT1 at Tyr701 were analyzed by immunoblotting analysis. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. b After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were treated with or without IFN-γ plus TNF-α (10 ng/ml each) for 6 h. Nucleus protein of cells was harvested and NF-κB p65 was analyzed by immunoblotting analysis. c After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, cells were harvested and IκBα expression was analyzed by immunoblotting analysis. d Control MSCs, vector-MSCs and UCHL1-MSCs were harvested, and IκBα expression was analyzed by immunoblotting analysis. e After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were pretreated with NF-κB inhibitor PDTC (1 μM) or STAT1 inhibitor Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (10 ng/ml each) treatment for 24 h. Cells were harvested and iNOS expression was analyzed by immunoblotting analysis. f , g After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, irradiated untreated, DMSO-pretreated or LDN57444-pretreated murine MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with CFSE-labeled splenocytes for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P
    Figure Legend Snippet: UCHL1 inhibition promoted the activation of NF-κB and STAT1 signaling. a After pretreatment with DMSO or LDN57444 (10 μM), murine MSCs were treated with IFN-γ plus TNF-α (10 ng/ml each) for the indicated time. Cells were harvested and NF-κB p65, STAT1, phosphorylation of NF-κB p65 and STAT1 at Tyr701 were analyzed by immunoblotting analysis. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. b After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were treated with or without IFN-γ plus TNF-α (10 ng/ml each) for 6 h. Nucleus protein of cells was harvested and NF-κB p65 was analyzed by immunoblotting analysis. c After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, cells were harvested and IκBα expression was analyzed by immunoblotting analysis. d Control MSCs, vector-MSCs and UCHL1-MSCs were harvested, and IκBα expression was analyzed by immunoblotting analysis. e After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, murine MSCs were pretreated with NF-κB inhibitor PDTC (1 μM) or STAT1 inhibitor Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (10 ng/ml each) treatment for 24 h. Cells were harvested and iNOS expression was analyzed by immunoblotting analysis. f , g After pretreatment with DMSO or LDN57444 (10 μM) for 24 h, irradiated untreated, DMSO-pretreated or LDN57444-pretreated murine MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with CFSE-labeled splenocytes for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P

    Techniques Used: Inhibition, Activation Assay, Software, Expressing, Plasmid Preparation, Irradiation, Co-Culture Assay, Labeling, Flow Cytometry, Cytometry

    UCHL1 inhibition promoted the immunosuppressive capacity and IDO expression of human MSCs. a Human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 12 and 24 h, and UCHL1 expression was examined by immunoblotting analysis. b Human MSCs were pretreated with DMSO or LDN57444 (20 μM) for 24 h, and irradiated untreated, DMSO-pretreated or LDN57444-pretreated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the indicated ratios. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. c After pretreatment with DMSO or LDN57444 (20 μM or 30 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 24 h, and IDO expression was examined by immunoblotting analysis. d After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1, and 1-MT was added to block IDO. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. e After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each), phosphorylation of NF-κB p65 and STAT1 at Tyr701 were examined by immunoblotting analysis at the indicated time points. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. f After pretreatment with DMSO or LDN57444 (20 μM), human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (5 ng/ml each) treatment for 24 h, and IDO expression was examined by immunoblotting assay. g , h After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P
    Figure Legend Snippet: UCHL1 inhibition promoted the immunosuppressive capacity and IDO expression of human MSCs. a Human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 12 and 24 h, and UCHL1 expression was examined by immunoblotting analysis. b Human MSCs were pretreated with DMSO or LDN57444 (20 μM) for 24 h, and irradiated untreated, DMSO-pretreated or LDN57444-pretreated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the indicated ratios. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. c After pretreatment with DMSO or LDN57444 (20 μM or 30 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each) for 24 h, and IDO expression was examined by immunoblotting analysis. d After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were co-cultured with CFSE-labeled human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1, and 1-MT was added to block IDO. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. e After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, human MSCs were treated with human IFN-γ plus TNF-α (5 ng/ml each), phosphorylation of NF-κB p65 and STAT1 at Tyr701 were examined by immunoblotting analysis at the indicated time points. The densitometry of p-NF-κB p65 and p-STAT1 (Tyr701) was quantified using ImageJ software, and NF-κB p65 and STAT1 were used as controls, respectively. f After pretreatment with DMSO or LDN57444 (20 μM), human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to IFN-γ plus TNF-α (5 ng/ml each) treatment for 24 h, and IDO expression was examined by immunoblotting assay. g , h After pretreatment with DMSO or LDN57444 (20 μM) for 24 h, irradiated human MSCs were pretreated with PDTC (1 μM) or Fludarabine (2 μM) for 6 h prior to co-culture with human PBMCs for 3 days in the presence of anti-CD3/CD28 antibodies at the ratio of 40: 1. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P

    Techniques Used: Inhibition, Expressing, Irradiation, Cell Culture, Labeling, Flow Cytometry, Cytometry, Co-Culture Assay, Blocking Assay, Software

    6) Product Images from "Regulatory effects of the JAK3/STAT1 pathway on the release of secreted phospholipase A2-IIA in microvascular endothelial cells of the injured brain"

    Article Title: Regulatory effects of the JAK3/STAT1 pathway on the release of secreted phospholipase A2-IIA in microvascular endothelial cells of the injured brain

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-9-170

    Effects of L-NAME, AG, WHI, Flu, and STAT1 siRNA on the phosphorylation of STAT1 and nuclear STAT1 expression in BMVECs after LPS treatment. ( A ) Effects of L-NAME, AG, WHI, and Flu on the phosphorylation of STAT1 Tyr701 in BMVECs were detected using Western blotting. The data are presented as the means ± SE of four separate experiments. * P
    Figure Legend Snippet: Effects of L-NAME, AG, WHI, Flu, and STAT1 siRNA on the phosphorylation of STAT1 and nuclear STAT1 expression in BMVECs after LPS treatment. ( A ) Effects of L-NAME, AG, WHI, and Flu on the phosphorylation of STAT1 Tyr701 in BMVECs were detected using Western blotting. The data are presented as the means ± SE of four separate experiments. * P

    Techniques Used: Expressing, Western Blot

    7) Product Images from "Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway"

    Article Title: Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1745094

    NS4A interacts with the SH2 and TAD domain of STAT1 or STAT2 to impair their phosphorylation. (A) In vitro kinase assay of STAT1 phosphorylated at Tyr701 (Y701) and STAT2 phosphorylated at Tyr690 (Y690). The peptide of STAT1 or STAT2 and their kinase JAK1 or TYK2 were introduced into a mixture containing Flag-NS4A. (B) Schematic representation of STAT1 or STAT2 (top). IB analysis of HEK293 T cells co-transfected for 48 h with Flag-NS4A and HA-wild-type STAT1 or STAT2, or their mutants, assessed with whole-cell lysates (10% input) or IP with anti-HA antibody (bottom). The numbers in A means the relative intensity of the p-STAT1/ STAT1 or p-STAT2/STAT2 detected by Western blotting. Data are representative of three independent experiments.
    Figure Legend Snippet: NS4A interacts with the SH2 and TAD domain of STAT1 or STAT2 to impair their phosphorylation. (A) In vitro kinase assay of STAT1 phosphorylated at Tyr701 (Y701) and STAT2 phosphorylated at Tyr690 (Y690). The peptide of STAT1 or STAT2 and their kinase JAK1 or TYK2 were introduced into a mixture containing Flag-NS4A. (B) Schematic representation of STAT1 or STAT2 (top). IB analysis of HEK293 T cells co-transfected for 48 h with Flag-NS4A and HA-wild-type STAT1 or STAT2, or their mutants, assessed with whole-cell lysates (10% input) or IP with anti-HA antibody (bottom). The numbers in A means the relative intensity of the p-STAT1/ STAT1 or p-STAT2/STAT2 detected by Western blotting. Data are representative of three independent experiments.

    Techniques Used: In Vitro, Kinase Assay, Transfection, Western Blot

    8) Product Images from "An Intracytoplasmic IL-10 Receptor Variant Permits Rapid Reduction in STAT3 Activation"

    Article Title: An Intracytoplasmic IL-10 Receptor Variant Permits Rapid Reduction in STAT3 Activation

    Journal: Genes and immunity

    doi: 10.1038/gene.2011.12

    Dynamics of STAT3, STAT1 and JAK1 phosphorylation in HeLa cells expressing wildtype or mutant IL10R1 HeLa cells transfected with IL10R1-WT,-SNP3, -SNP4 or –SNP3+4 were cultured in the absence (−) or presence of IL-10 (IL-10 cont.), or treated with an IL-10 pulse for 30 min (IL-10 pulse). After 0.5, 3, 6, 9, 12 and 24h, cells were lysed and phosphorylation of STAT3 at Tyr705 (a) and STAT1 at Tyr701 (b) was detected by western blot. (c) JAK1 phosphorylation was detected after 6h (6h IL-10 or 30min IL-10 pulse).
    Figure Legend Snippet: Dynamics of STAT3, STAT1 and JAK1 phosphorylation in HeLa cells expressing wildtype or mutant IL10R1 HeLa cells transfected with IL10R1-WT,-SNP3, -SNP4 or –SNP3+4 were cultured in the absence (−) or presence of IL-10 (IL-10 cont.), or treated with an IL-10 pulse for 30 min (IL-10 pulse). After 0.5, 3, 6, 9, 12 and 24h, cells were lysed and phosphorylation of STAT3 at Tyr705 (a) and STAT1 at Tyr701 (b) was detected by western blot. (c) JAK1 phosphorylation was detected after 6h (6h IL-10 or 30min IL-10 pulse).

    Techniques Used: Expressing, Mutagenesis, Transfection, Cell Culture, Western Blot

    9) Product Images from "The synergistic interaction between the calcineurin B subunit and IFN-γ enhances macrophage antitumor activity"

    Article Title: The synergistic interaction between the calcineurin B subunit and IFN-γ enhances macrophage antitumor activity

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.92

    CnB stimulates phosphorylation of STAT1, and combination treatment with CnB and IFN- γ markedly enhances phosphorylation at Ser727 but not at Tyr701. ( a ) Western blotting detection of time-dependent phosphorylation of STAT1. RAW264.7 cells were treated with 20 μ g/ml CnB for 0-300 min. ( b ) Proteinase K-digested CnB loses the ability to induce phosphorylation of STAT1 at Ser727. Cells were incubated with CnB (1–40 μ g/ml) or proteinase K-digested CnB (1–40 μ g/ml) for 25 min. ( c ) CnB pretreatment enhances IFN- γ -induced phosphorylation of STAT1 at Ser727 but not at Tyr701 (time course). Cells were preincubated with or without CnB (20 μ g/ml) for 20 min, followed by incubation with IFN- γ (1 ng/ml) for 3–25 min. ( d ) Combined treatment with CnB and IFN- γ synergistically promotes STAT1 phosphorylation at Ser727 but not at Tyr701 (dose course). Duration of treatments, 25 min. Phosphorylation levels of STAT1 were quantified by densitometry and normalized to total STAT1 expression ( Supplementary Figure S1 )
    Figure Legend Snippet: CnB stimulates phosphorylation of STAT1, and combination treatment with CnB and IFN- γ markedly enhances phosphorylation at Ser727 but not at Tyr701. ( a ) Western blotting detection of time-dependent phosphorylation of STAT1. RAW264.7 cells were treated with 20 μ g/ml CnB for 0-300 min. ( b ) Proteinase K-digested CnB loses the ability to induce phosphorylation of STAT1 at Ser727. Cells were incubated with CnB (1–40 μ g/ml) or proteinase K-digested CnB (1–40 μ g/ml) for 25 min. ( c ) CnB pretreatment enhances IFN- γ -induced phosphorylation of STAT1 at Ser727 but not at Tyr701 (time course). Cells were preincubated with or without CnB (20 μ g/ml) for 20 min, followed by incubation with IFN- γ (1 ng/ml) for 3–25 min. ( d ) Combined treatment with CnB and IFN- γ synergistically promotes STAT1 phosphorylation at Ser727 but not at Tyr701 (dose course). Duration of treatments, 25 min. Phosphorylation levels of STAT1 were quantified by densitometry and normalized to total STAT1 expression ( Supplementary Figure S1 )

    Techniques Used: Western Blot, Incubation, Expressing

    CnB-induced STAT1 phosphorylation is dependent on the integrin α M–p38 signaling pathway. ( a ) CnB promotes phosphorylation of p38 in a time-dependent manner. RAW264.7 macrophages were incubated with 20 μ g/ml CnB for 0–300 min, and then lysed for western blotting analysis. ( b ) The p38 inhibitor SB203580 markedly inhibits CnB-induced STAT1 Ser727 and Tyr701 phosphorylation. Cells were pretreated with SB203580 (1–50 μ M) for 30 min followed by CnB/IFN- γ treatment for another 30 min. ( c ) CnB-induced p38 phosphorylation is blocked by integrin α M antibody. Antibody concentrations, 7.5 or 15 μ g/ml; duration of antibody pretreatment, 45 min. ( d ) CnB-induced p38 phosphorylation is attenuated by integrin α M knockdown. Forty-eight hours after siRNAs transfection, cells were incubated with 5 μ g/ml CnB for 25 min. ( e ) CnB-induced STAT1 phosphorylation is attenuated by integrin α M knockdown. Forty-eight hours after siRNAs transfection, cells were incubated with CnB (5 μ g/ml) or IFN- γ (1 ng/ml) for 25 min. Phosphorylation levels of STAT1 and p38 were quantified by densitometry and normalized to total STAT1 or total p38 expression, respectively ( Supplementary Figure S2 )
    Figure Legend Snippet: CnB-induced STAT1 phosphorylation is dependent on the integrin α M–p38 signaling pathway. ( a ) CnB promotes phosphorylation of p38 in a time-dependent manner. RAW264.7 macrophages were incubated with 20 μ g/ml CnB for 0–300 min, and then lysed for western blotting analysis. ( b ) The p38 inhibitor SB203580 markedly inhibits CnB-induced STAT1 Ser727 and Tyr701 phosphorylation. Cells were pretreated with SB203580 (1–50 μ M) for 30 min followed by CnB/IFN- γ treatment for another 30 min. ( c ) CnB-induced p38 phosphorylation is blocked by integrin α M antibody. Antibody concentrations, 7.5 or 15 μ g/ml; duration of antibody pretreatment, 45 min. ( d ) CnB-induced p38 phosphorylation is attenuated by integrin α M knockdown. Forty-eight hours after siRNAs transfection, cells were incubated with 5 μ g/ml CnB for 25 min. ( e ) CnB-induced STAT1 phosphorylation is attenuated by integrin α M knockdown. Forty-eight hours after siRNAs transfection, cells were incubated with CnB (5 μ g/ml) or IFN- γ (1 ng/ml) for 25 min. Phosphorylation levels of STAT1 and p38 were quantified by densitometry and normalized to total STAT1 or total p38 expression, respectively ( Supplementary Figure S2 )

    Techniques Used: Incubation, Western Blot, Transfection, Expressing

    Diagram illustrating the mechanism of synergism between CnB and IFN-γ. CnB binds to an important macrophage membrane receptor integrin α M and activate p38 MAPK. Activated p38 promotes strong phosphorylation of STAT1 at Ser727 and weak phosphorylation at Tyr701. At the same time, IFN- γ binds to IFN- γ receptors (IFNGR) and strongly induces STAT1 Ser727 phosphorylation by p38 (or PKC- δ ) and STAT1 Tyr701 phosphorylation by Jak2. Combination treatment with CnB and IFN- γ greatly enhances STAT1 serine and tyrosine phosphorylation and activates STAT1 maximally. The immense activation of STAT1 causes a sudden burst of TRAIL and other antitumor M1 cytokines directly or via the transcriptional factors IRF-1 and IRF-9 and finally render macrophages re-educated, possessing more ability to eradicate tumor. ‘+++' represents strong activation and ‘+' represents weak activation
    Figure Legend Snippet: Diagram illustrating the mechanism of synergism between CnB and IFN-γ. CnB binds to an important macrophage membrane receptor integrin α M and activate p38 MAPK. Activated p38 promotes strong phosphorylation of STAT1 at Ser727 and weak phosphorylation at Tyr701. At the same time, IFN- γ binds to IFN- γ receptors (IFNGR) and strongly induces STAT1 Ser727 phosphorylation by p38 (or PKC- δ ) and STAT1 Tyr701 phosphorylation by Jak2. Combination treatment with CnB and IFN- γ greatly enhances STAT1 serine and tyrosine phosphorylation and activates STAT1 maximally. The immense activation of STAT1 causes a sudden burst of TRAIL and other antitumor M1 cytokines directly or via the transcriptional factors IRF-1 and IRF-9 and finally render macrophages re-educated, possessing more ability to eradicate tumor. ‘+++' represents strong activation and ‘+' represents weak activation

    Techniques Used: Activation Assay

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    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 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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    Cell Signaling Technology Inc stat1
    PG102 inhibits NF-κB and <t>STAT1</t> signaling in HaCaT cells. ( A ) Western blot results of corresponding proteins after treatment with M5 and PG102 for 30 min. ( B ) Densitometry of Western blot. The means of three experiments are shown. ### p
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    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).
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    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

    PG102 inhibits NF-κB and STAT1 signaling in HaCaT cells. ( A ) Western blot results of corresponding proteins after treatment with M5 and PG102 for 30 min. ( B ) Densitometry of Western blot. The means of three experiments are shown. ### p

    Journal: Nutrients

    Article Title: A Water-Soluble Extract from Actinidia arguta Ameliorates Psoriasis-Like Skin Inflammation in Mice by Inhibition of Neutrophil Infiltration

    doi: 10.3390/nu10101399

    Figure Lengend Snippet: PG102 inhibits NF-κB and STAT1 signaling in HaCaT cells. ( A ) Western blot results of corresponding proteins after treatment with M5 and PG102 for 30 min. ( B ) Densitometry of Western blot. The means of three experiments are shown. ### p

    Article Snippet: The membrane was then incubated with antibodies specific for phospho-STAT3 (#9134, #9145), STAT1 (#9167), p65 (#3033), and STAT3 (#4904), STAT1 (#9172), p65 (#8242), IκB-α (#9242) (1:1000; Cell Signaling Technology, Danvers, MA, USA), and β-actin (A5441, Sigma-Aldrich) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1:100,000; Sigma-Aldrich) at room temperature for 1 h. The blot was developed by Immobilon ECL HRP substrate (Merck) and visualized by exposure on autoradiography film.

    Techniques: Western Blot

    Species-specific activity of rec Mg IFN-γ and mouse IFN-γ on different rodent cell lines. NIH/3T3, BVK168, FMN-R and AAL-R cells were tested and either left untreated as control (lower panel), stimulated for 1 h with rec Mg IFN-γ (200 ng/ml, upper panel) or mouse IFN-γ (200 U/ml, central panel). In each panel the following stainings are shown from left to right: phospho-Tyr701-STAT1 staining (green), DAPI staining (white) and overlay of the two signals. Activated phospho-STAT1 is indicated by nuclear translocation. Of note, minor background signal in the cytoplasma was observed in some control treated cells. A representative experiment of three replicates is shown. The scale bar represents 50 μm.

    Journal: Scientific Reports

    Article Title: Recombinant IFN-γ from the bank vole Myodes glareolus: a novel tool for research on rodent reservoirs of zoonotic pathogens

    doi: 10.1038/s41598-018-21143-0

    Figure Lengend Snippet: Species-specific activity of rec Mg IFN-γ and mouse IFN-γ on different rodent cell lines. NIH/3T3, BVK168, FMN-R and AAL-R cells were tested and either left untreated as control (lower panel), stimulated for 1 h with rec Mg IFN-γ (200 ng/ml, upper panel) or mouse IFN-γ (200 U/ml, central panel). In each panel the following stainings are shown from left to right: phospho-Tyr701-STAT1 staining (green), DAPI staining (white) and overlay of the two signals. Activated phospho-STAT1 is indicated by nuclear translocation. Of note, minor background signal in the cytoplasma was observed in some control treated cells. A representative experiment of three replicates is shown. The scale bar represents 50 μm.

    Article Snippet: For the immunofluorescent analysis a rabbit anti-phospho-STAT1 (Tyr701) monoclonal antibody (dilution 1:50) or rabbit polyclonal anti-phospho-STAT1 (Ser727) antibody (dilution 1:100; both Cell Signaling Technology) and an AlexaFluor 488-conjugated goat anti-rabbit secondary antibody (dilution 1:1,000; Abcam) were used.

    Techniques: Activity Assay, Staining, Translocation Assay

    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).

    Journal: Neuro-Oncology

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

    doi: 10.1093/neuonc/nox051

    Figure Lengend 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).

    Article Snippet: Antibodies to human CD45 (clone 2B11) was from DakoCytomation, STAT1 and phosphorylated STAT1 (pSTAT1; Tyr701) were purchased from Cell Signaling, phycoerythrin-labeled anti-IFNAR2 from PBL Assay Science, and anti–MHC-I (clone W/32) and anti–human leukocyte antigen (HLA)-DR (clone L243) were kindly provided by S. Stevanović (Tübingen, Germany).

    Techniques: 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.

    Journal: Neuro-Oncology

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

    doi: 10.1093/neuonc/nox051

    Figure Lengend 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.

    Article Snippet: Antibodies to human CD45 (clone 2B11) was from DakoCytomation, STAT1 and phosphorylated STAT1 (pSTAT1; Tyr701) were purchased from Cell Signaling, phycoerythrin-labeled anti-IFNAR2 from PBL Assay Science, and anti–MHC-I (clone W/32) and anti–human leukocyte antigen (HLA)-DR (clone L243) were kindly provided by S. Stevanović (Tübingen, Germany).

    Techniques: 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.

    Journal: Neuro-Oncology

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

    doi: 10.1093/neuonc/nox051

    Figure Lengend 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.

    Article Snippet: Antibodies to human CD45 (clone 2B11) was from DakoCytomation, STAT1 and phosphorylated STAT1 (pSTAT1; Tyr701) were purchased from Cell Signaling, phycoerythrin-labeled anti-IFNAR2 from PBL Assay Science, and anti–MHC-I (clone W/32) and anti–human leukocyte antigen (HLA)-DR (clone L243) were kindly provided by S. Stevanović (Tübingen, Germany).

    Techniques: 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).

    Journal: Neuro-Oncology

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

    doi: 10.1093/neuonc/nox051

    Figure Lengend 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).

    Article Snippet: Antibodies to human CD45 (clone 2B11) was from DakoCytomation, STAT1 and phosphorylated STAT1 (pSTAT1; Tyr701) were purchased from Cell Signaling, phycoerythrin-labeled anti-IFNAR2 from PBL Assay Science, and anti–MHC-I (clone W/32) and anti–human leukocyte antigen (HLA)-DR (clone L243) were kindly provided by S. Stevanović (Tübingen, Germany).

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