anti py stat1  (Cell Signaling Technology Inc)

Journal: Oncology letters

Article Title: Machine learning model reveals roles of interferon‑stimulated genes in sorafenib‑resistant liver cancer.

doi: 10.3892/ol.2024.14571

Figure Lengend Snippet: Figure 2. Increased IRF9 expression in sorafenib‑resistant liver cancer cells. (A) Procedure for establishing sorafenib‑resistant liver cancer cells. (B) liver cancer cells were treated with an increasing dose of sorafenib for 24 h. Cell viability was measured by MTT assay. (C) Protein levels of STAT1, STAT2 and IRF9 from immunoblotting. (D) mRNA levels of STAT1, STAT2 and IRF9 from reverse transcription‑quantitative PCR. *P<0.05 and **P<0.01 vs. liver cancer cell lines (Huh‑7 and HepG2). IRF9, interferon regulatory factor 9; wks, weeks; p‑, phosphorylated.

Article Snippet: After blocking the membrane in TBS containing 5% skim milk for 1 h. The antibodies used for immunoblotting were as follows: rabbit monoclonal anti‐STAT1 (Cell signaling Technology, Cat#9176S), rabbit monoclonal anti‐PY STAT1 (Cell signaling Technology, Cat#9167S), rabbit polyclonal anti‐STAT2 (Cell signaling Technology, Cat#4594S), rabbit polyclonal anti‐PY STAT2 (Cell signaling Technology, Cat#4441S), rabbit monoclonal IRF9 (Cell signaling Technology, Cat#28492), and horseradish peroxidase‐conjugated secondary antibody (1:5,000). siRNA transfection.

Techniques: Expressing, MTT Assay, Western Blot

Journal: Oncology letters

Article Title: Machine learning model reveals roles of interferon‑stimulated genes in sorafenib‑resistant liver cancer.

doi: 10.3892/ol.2024.14571

Figure Lengend Snippet: Figure 4. U‑ISGs unresponsiveness depends on STAT1, STAT2 and IRF9 in Huh‑7‑SR cells. (A) Huh‑7‑SR cells were transfected with si‑control, si‑STAT1, si‑STAT2, and si‑IRF9. Then, 48 h after transfection, cells were harvested and immunoblotting of STAT1, STAT2 and IRF9 was performed. (B) mRNA levels of U‑ISGs were measured by reverse transcription‑quantitative PCR. (C) After transfection, Huh‑7‑SR cells were treated with an increasing dose of sorafenib for 24 h. **P<0.01 vs. siControl. IRF, interferon regulatory factor; si, small interfering; OAS1; oligoadenylate synthetase 1; IFI27, Interferon Alpha Inducible Protein 27.

Article Snippet: After blocking the membrane in TBS containing 5% skim milk for 1 h. The antibodies used for immunoblotting were as follows: rabbit monoclonal anti‐STAT1 (Cell signaling Technology, Cat#9176S), rabbit monoclonal anti‐PY STAT1 (Cell signaling Technology, Cat#9167S), rabbit polyclonal anti‐STAT2 (Cell signaling Technology, Cat#4594S), rabbit polyclonal anti‐PY STAT2 (Cell signaling Technology, Cat#4441S), rabbit monoclonal IRF9 (Cell signaling Technology, Cat#28492), and horseradish peroxidase‐conjugated secondary antibody (1:5,000). siRNA transfection.

Techniques: Transfection, Western Blot

Journal: PLoS Pathogens

Article Title: Definition of the immune evasion-replication interface of rabies virus P protein

doi: 10.1371/journal.ppat.1009729

Figure Lengend Snippet: (A) COS-7 cells co-transfected to express the indicated proteins were treated 24 h post-transfection with or without IFN-α (1000 U/ml, 30 min) before lysis and immunoprecipitation for GFP. Lysates (input) and immunoprecipitates (IP) were analysed by immunoblotting (IB) using antibodies against the indicated proteins. Results are representative of 3 independent assays and show data from a single blot with intervening and marker lanes removed. (B) HEK-293T cells co-transfected to express the indicated proteins were treated 24 h post-transfection with or without IFN-α (1000 U/ml, 1 h) before lysis, immunoprecipitation for GFP and IB, as above. Results are representative of 2 independent assays. (C) COS-7 cells co-transfected to express the indicated proteins were treated 24 h post-transfection with or without IFN-α (1000 U/ml, 30 min) before fixation, immunofluorescent staining for STAT1 (blue) and analysis by confocal laser scanning microscopy. Representative images are shown. Arrowheads indicate cells with detectable expression of the transfected GFP- and mCherry-fused proteins. (D) Images such as those shown in (C) were analysed to calculate the Fn/c for GFP, mCherry, and immunostained STAT1 (mean ± SEM; n ≥ 43 cells for each condition; results are from a single assay representative of two independent assays). Statistical analysis used Student’s t test. ****, p < 0.0001; NS, not significant.

Article Snippet: The lysate (input) and IP samples were separated by SDS-PAGE before analysis by western blot using antibodies for GFP (Roche Applied Science, catalog no. 11814460001), mCherry (Abnova, catalog no. PAB18013 or Abcam, catalog no. ab167453), FLAG (Sigma-Aldrich, catalog no. F1804), STAT1 (BD Biosciences, catalog no. 610185 or Cell Signaling Technology, catalog no. 14994) or pY-STAT1 (Cell Signaling Technology, catalog no. 9176).

Techniques: Transfection, Lysis, Immunoprecipitation, Western Blot, Marker, Staining, Confocal Laser Scanning Microscopy, Expressing

Journal: Nature immunology

Article Title: Activation of naïve CD4 + T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4 + T cells

doi: 10.1038/s41590-019-0350-0

Figure Lengend Snippet: (a) Representative H&E staining of knee joints at day 10 post disease induction (antigen-induced arthritis, AIA) (bar: 500μm); boxed area shows the location of the immunofluorescence. Representative immunofluorescence with antibodies against CD3 (red), pY-STAT1 or pY-STAT3 (green) is shown together with DAPI counterstaining (blue) (bar: 100μm). Graph shows the proportion of CD3 + T cells displaying either pY STAT1 or pY-STAT3 (n=3). (b) Phosphorylation of STAT1 and STAT3 by flow cytometry of infiltrating synovial CD4 + T cells during AIA after stimulation with 20ng/ml IL-6 compare to CD4 + T N cells. (c) Representative flow cytometry of pY-STAT1 and pY-STAT3 in CD4 + T cells extracted from inguinal lymph nodes of mBSA challenged (n=4) and non-challenged mice (control) (n=3) following stimulation with 20ng/ml IL-6 for 30 min. Graphs show quantification of pY-STAT1 and pY-STAT3 activity in CD4 + T N and CD4 + T EM cells (n=4). (d) Quantitative PCR of Ahr, Ifng, Il17a, Il21, Rorc, Socs3 and Stat3 in CD4 + T N (n=4) and CD4 + T EM cells (n=2) extracted from inguinal lymph nodes of mBSA challenged mice. (e) Intracellular flow cytometry analysis of IL-21 production in CD4 + T N and CD4 + T EM cells extracted from inguinal lymph nodes after 4 hours stimulation with PMA, ionomycin and monensin (n=4). Data are representative of three independent experiments (c,e), two independent experiments (a,b) and one experiment involving biological replicates (d). **** P <0.0001, ** P <0.01, * P <0.05 (Two-tailed unpaired Student’s t test (a,b,d,e) and one-way ANOVA test with Tukey’s multiple comparison test (c). Data are shown as mean ± s.d.)

Article Snippet: For immunohistochemistry, antigens were detected in paraffin sections using antibodies against CD3 (A0452, Dako), pY-STAT1 (Tyr701; 58D6) and PTPN2 (AF1930, R&D Systems).

Techniques: Staining, Immunofluorescence, Phospho-proteomics, Flow Cytometry, Control, Activity Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, Comparison

Journal: Nature immunology

Article Title: Activation of naïve CD4 + T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4 + T cells

doi: 10.1038/s41590-019-0350-0

Figure Lengend Snippet: (a) Representative flow cytometry analysis of STAT1 and STAT3 responses in naïve (T N ), central memory (T CM ), effector (T Eff ) and effector memory (T EM ) CD4 + T cells after 30 min IL-6 stimulation (20ng/ml). Numbers indicate the percentage of pY-STAT1 or pY STAT3 staining. Temporal changes in pY-STAT1 and pY-STAT3 are shown for each T cell subset following IL 6 stimulation (n=3). (b) Detection of pY-STAT1 and pY-STAT3 in CD4 + T N , CD4 + T CM , CD4 + T Eff and CD4 + T EM cells from WT and IL6ra -/- mice. CD4 + T cells were stimulated for 30 min with an equimolar concentration of IL-6 or an IL-6-sIL-6R fusion protein (HDS) (n=3). (c) Intracellular flow cytometry analysis of pY-STAT1 in CD4 + T cells following 30 min stimulation with IL-6, IL-27 or IFNγ (20ng/ml) (n=3). (d) Microarray expression data is presented for CD4 + T N (n=3), CD4 + T EM (n=3), and in vitro expanded CD4 + effector-like T cells (See , CD4 + T EXP ) (n=4) treated with 20ng/ml IL-6 for 6 hours. Analysis was confined to genes displaying both a relative signal intensity of >150 and >1.5-fold alteration in expression following IL-6 treatment ( P <0.05). Heat map is hierarchically clustered based in the relative expression (Z-score) (left panel) or Fold change (right panel). (e) Volcano plots displaying IL-6 regulated gene expression in CD4 + T N and CD4 + T EXP cells stimulated with IL 6 (20ng/ml) or in combination with antibodies against CD3 and CD28. An interactive figure can be found on-line ( http://jones-cytokinelab.co.uk/NI2019/figure2d.shtml ). Data are representative of two independent experiments (a,c) and one experiment involving biological replicates (b,d,e). *** P <0.001 * P <0.05 (Two-tailed unpaired Student’s t test (a) and one-way ANOVA with Tukey’s multiple comparison test (b,c). Data are shown as mean ± s.e.m).

Article Snippet: For immunohistochemistry, antigens were detected in paraffin sections using antibodies against CD3 (A0452, Dako), pY-STAT1 (Tyr701; 58D6) and PTPN2 (AF1930, R&D Systems).

Techniques: Flow Cytometry, Staining, Concentration Assay, Microarray, Expressing, In Vitro, Gene Expression, Two Tailed Test, Comparison

Journal: Nature immunology

Article Title: Activation of naïve CD4 + T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4 + T cells

doi: 10.1038/s41590-019-0350-0

Figure Lengend Snippet: (a) CD4 + T N and CD4 + T EM cells were pre-treated for 5 min with 5mM sodium orthovanadate (vanadate) prior to IL-6 (20ng/ml) stimulation for 30 min. Changes in pY-STAT1 and pY-STAT3 activity were monitored by intracellular flow cytometry (MFI). A comparable analysis of pS-STAT1 and pS-STAT3 is shown as a control (n=3). (b) Quantitative PCR for Ahr , Il21 , Stat3 and Socs3 after vanadate pre-treatment and 20ng/ml IL-6 stimulation in CD4 + T EM cells (n=3). (c) Heatmap analysis of Affymetrix transcriptomic data identifies the top 20 genes ( P <0.05; relative signal intensity of >150; 1.5-fold alteration) associated with protein tyrosine phosphatase enzyme family. Data is presented as a hierarchical cluster using the average linkage method (row 1-pearson rank correlation). Data are representative of two independent experiment (a,b) and one experiment involving biological replicates (c). *** P <0.001; ** P <0.01 (one-way ANOVA with Tukey’s multiple comparison test (a) and two-way ANOVA with Sidak multiple comparison test (b). Data are shown as mean ± s.e.m (a) and mean ± s.d (b).

Article Snippet: For immunohistochemistry, antigens were detected in paraffin sections using antibodies against CD3 (A0452, Dako), pY-STAT1 (Tyr701; 58D6) and PTPN2 (AF1930, R&D Systems).

Techniques: Activity Assay, Flow Cytometry, Control, Real-time Polymerase Chain Reaction, Comparison

Journal: Nature immunology

Article Title: Activation of naïve CD4 + T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4 + T cells

doi: 10.1038/s41590-019-0350-0

Figure Lengend Snippet: ( a ) Representative histogram of PTPN2 staining in CD4 + T N and CD4 + T EM cells by flow cytometry. ( b ) Flow cytometry analysis of STAT1 phosphorylation and PTPN2 expression in CD4 + T N and CD4 + T EM cells analyzed 30 min after stimulation with 20 ng/ml IL-6. ( c ) Immunohistochemistry of the inflamed synovium from wild-type mice with antigen-induced arthritis (day-10 post disease induction) in tissue sections stained with antibodies against CD3, Ptpn2 and pY STAT1. Scale bar, 100μm (left panel) and 200μm (right panel). ( d ) Analysis of pY-STAT1 and pY-STAT3 in CD4 + T N and CD4 + T EXP cells derived from Ptpn2 fl/fl , Lck-Cre Ptpn2 fl/fl (left panel) or wild-type and Ptpn22 -/- mice (right panel) (n=4) exposed to IL-6 (20 ng/ml) for 30 min in combination with antibodies against CD3 and CD28. Fold change relative to the untreated controls are compared. ( e ) IL-21 and IL-17A quantification by flow cytometry in CD4 + T EM cells from Ptpn2 fl/fl and Lck-Cre Ptpn2 fl/fl mice (n=3). (f) ImageStream analysis of STAT1 and PTPN2 localization in CD4 + T N and CD4 + T EM cells stained with antibodies against STAT1, pY-STAT1, PTPN2 and CD4. Data are representative of three independent experiments (a,b), two independent experiments (f) and one experiment involving biological replicates (c,d,e). **** P <0.0001; *** P <0.001 (One-way ANOVA with Tukey’s multiple comparison test (d) and Two-tailed unpaired Student’s test (e). Data are shown as mean ± s.d.).

Article Snippet: For immunohistochemistry, antigens were detected in paraffin sections using antibodies against CD3 (A0452, Dako), pY-STAT1 (Tyr701; 58D6) and PTPN2 (AF1930, R&D Systems).

Techniques: Staining, Flow Cytometry, Phospho-proteomics, Expressing, Immunohistochemistry, Derivative Assay, Comparison, Two Tailed Test

Journal: Nature immunology

Article Title: Activation of naïve CD4 + T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4 + T cells

doi: 10.1038/s41590-019-0350-0

Figure Lengend Snippet: (a) Circos visualisation details the IL-6 regulated gene changes in CD4 + T N and CD4 + T EXP cells (See ), and ex vivo sorted CD4 + T EM cells. Total number of IL-6 regulated genes is presented in parenthesis for each population ( P < 0.05, Chip Intensity 150+, and > 1.5-fold change). Lines coloured in red represent up-regulated genes and all down-regulated gene changes are blue. Connecting lines highlight common genes that are IL-6 regulated in two or more of the populations. (b) IPA analysis of genes associated with IL-6, STAT1 and STAT3 upstream regulators. Top left heat map shows the predicted activated state (orange) and the predicted inhibited state (blue) of transcription regulators. Upstream regulator analysis for CTLA4 and CD3 are presented as controls. Relative expression heat maps are presented as a hierarchical cluster using the average linkage method (row 1-pearson rank correlation). The differential expression of genes being regulated by IL-6, STAT1 or STAT3 is shown for CD4 + T N , CD4 + T EXP and CD4 + T EM cells. (c) IL-6 regulated gene changes derived from transcriptomic analysis were directly compared with datasets derived from IL-6 stimulated Stat1 -/- and Stat3 -/- CD4 + T cells (GSE65621).

Article Snippet: For immunohistochemistry, antigens were detected in paraffin sections using antibodies against CD3 (A0452, Dako), pY-STAT1 (Tyr701; 58D6) and PTPN2 (AF1930, R&D Systems).

Techniques: Ex Vivo, Expressing, Quantitative Proteomics, Derivative Assay

Journal: Nature immunology

Article Title: Activation of naïve CD4 + T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4 + T cells

doi: 10.1038/s41590-019-0350-0

Figure Lengend Snippet: ChIP-seq was performed on genomic DNA extracted from sorted CD4 + T N and CD4 + T EM cells following 1-hour stimulation with IL-6 in presence of antibodies against CD3 and CD28. Peak calling and downstream data processing are described in Materials & Methods . (a) Pie charts show the proportion of peaks associated with STAT1 and STAT3 binding to defined genomic regions. The total number of peaks identified is displayed graphically. All datasets residing outside TSS regions were only included if located to exonic or intronic sites. (b) Analysis of gene clusters regulated by binding STAT1 and STAT3 in TSS promoter regions. The heat map shows the score value for each gene identified with Homer for STAT1 and STAT3 ChIP-seq data in CD4 + T N (blue) and CD4 + T EM (red) cells. (c) Comparison of ChIP-seq datasets against Affymetrix gene expression (relative significance; -(log10 (adjusted P -value)). Analysis of STAT1 and STAT3 datasets is shown for CD4 + T N (blue) and CD4 + T EM (red) subsets. An interactive figure of additional information can be found on-line ( http://jones-cytokinelab.co.uk/NI2019/figure6c.shtml ) (d) Circos visualization of STAT1 and STAT3 binding to TSS regions of genes under IL-6 regulation in CD4 + T N and CD4 + T EM cells. Connecting lines are color coded to reflect involvement of STAT1 (green), STAT3 (blue) or both STAT1 and STAT3 (orange).

Article Snippet: For immunohistochemistry, antigens were detected in paraffin sections using antibodies against CD3 (A0452, Dako), pY-STAT1 (Tyr701; 58D6) and PTPN2 (AF1930, R&D Systems).

Techniques: ChIP-sequencing, Binding Assay, Comparison, Gene Expression

Journal: Nature immunology

Article Title: Activation of naïve CD4 + T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4 + T cells

doi: 10.1038/s41590-019-0350-0

Figure Lengend Snippet: (a) Circos plot shows the co-localisation of STAT1 (blue) and STAT3 (orange) binding to genomic regions sharing P300 enrichment in CD4 + T N and CD4 + T EM cells. The connecting lines show the relationship of STAT1 and STAT3 binding between CD4 + T N and CD4 + T EM cells. P300 ChIP-seq datasets (Accession number GSE40463, GSE60482) are derived from T H 1, T H 2 and T H 17 cells. (b) Heat map showing the expression of all IL-6 regulated genes linked with P300 binding in CD4 + T N and CD4 + T EM cells (positioned left). The correspondingly aligned heatmap (positioned right) shows the relationship to P300 sites in T H 1, T H 2 and T H 17 cells and shows the number of clustered P300 sites affiliated to an individual gene (blue=0, yellow=4). Specific examples of individual genes are shown. (c) Circos visualisation of 135 genes that display P300 binding in association with either STAT1 or STAT3 in CD4 + T N versus CD4 + T EM cells. (d) IPA predictions of the five distinct patterns of STAT binding identified from panel c. Hierarchical clustering of canonical pathways was performed using -Log ( P -value). lists the canonical pathways represented in the heatmap. (e) STAT1 binding enrichment quantification by ChIP-qPCR in Ptpn2 fl/fl and Lck-Cre:Ptpn2 fl/fl CD4 + T EM cells (one experiment with pool samples from 12 Ptpn2 fl/fl and 8 Lck-Cre Ptpn2 fl/fl mice).

Article Snippet: For immunohistochemistry, antigens were detected in paraffin sections using antibodies against CD3 (A0452, Dako), pY-STAT1 (Tyr701; 58D6) and PTPN2 (AF1930, R&D Systems).

Techniques: Binding Assay, ChIP-sequencing, Derivative Assay, Expressing, ChIP-qPCR

Journal: The Journal of Experimental Medicine

Article Title: MyD88 Primes Macrophages for Full-Scale Activation by Interferon-γ yet Mediates Few Responses to Mycobacterium tuberculosis

doi: 10.1084/jem.20030603

Figure Lengend Snippet: One potential mechanism of hyporesponsiveness of MyD88 −/− BMMφ to IFN-γ. (A) Western blot to detect phosphorylated STAT1 (pY-STAT1). WT and MyD88 −/− BMMφ were untreated or treated with 100 U/ml IFN-γ. Cell lysates were harvested 15 min and 2 h after IFN-γ treatment. Western blot was performed with anti-pTyr701 STAT1. (B) Hypothetical model for hyporesponsiveness of MyD88 −/− BMMφ to IFN-γ. The horizontal line represents the plasma membrane. Endogenous ligands that activate the MyD88 pathway might be produced by the macrophage (e.g., IL-1, IL-18, heat shock proteins) or might be produced by the action of macrophages on the extracellular matrix (e.g., fibronectin and fibronectin fragments). MyD88-dependent signaling in response to these ligands may drive the NF-κB–dependent expression of additional stimuli (e.g., TNF, SAA3), which can also help sustain NF-κB activation. The resulting activation of NF-κB in ostensibly resting macrophages can synergize with IFN-γ–activated signals (e.g., STAT1) in driving IFN-γ–dependent gene expression. (C) Detection of NF-κB by EMSA. Nuclear extracts were harvested from resting WT and MyD88 −/− macrophages and equal amounts of protein were subjected to EMSA. Data are representative of three experiments.

Article Snippet: The membrane was blocked with 5% milk, blotted with anti–mouse iNOS , or anti–mouse pY-STAT1 and anti–mouse STAT1 antibody (Cell Signaling Technology), followed by secondary antibody coupled to horseradish peroxidase (1:10,000; Amersham Biosciences).

Techniques: Western Blot, Produced, Expressing, Activation Assay