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, Flow Cytometry, 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, 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, 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, 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, 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, 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) 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

Journal: Frontiers in Immunology

Article Title: Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung

doi: 10.3389/fimmu.2016.00514

Figure Lengend Snippet: Socs1ΔNLS is localized in the cytoplasm . (A) NIH3T3 cells were transfected with the indicated eGFP-tagged plasmids and visualized by confocal microscopy. Nuclei were counterstained with Hoechst, and membranes were stained with CellMask™ dye. Scale bar, 5 μm. (B) NIH3T3 cells were transfected with eGFP-Socs1 and stimulated with IFNγ (50 ng/ml) as indicated. A z -stack was recorded and the ratio of the fluorescence in the cytoplasm versus the fluorescence in the nucleus was measured using ImageJ by setting an ROI around either the cytoplasm or the nucleus ( n = 3 with 20–50 cells each). Scale bar, 75 μm. (C) Western blot analysis of tyrosine phosphorylated STAT1. Raw264.7 cells were transfected with eGFP, eGFP-Socs1 , or eGFP-Socs1ΔNLS and stimulated with IFNγ (50 ng/ml) as indicated. Protein extracts were stained for pY-STAT1 (Tyr701), SOCS1, STAT1, and β-actin. Quantification using ImageJ is shown in (D) ( n = 3, mean + SD, two-way ANOVA including Bonferroni post-test).

Article Snippet: Blocking of unspecific binding was performed using 5% BSA solution in 1× TBST [1× TBS, 0.05% (v/v) Tween-20] for at least 1 h. Membranes were stained with antibodies against pY-STAT1 (Tyr701, #9167), STAT1 (#9172), IκBα (#9242), β-Actin (#4970) (Cell Signaling, Leiden, Netherlands; 1:1000), or hybridoma cell supernatant for SOCS1 detection (hybridoma cells newly generated by immunizing mice against the peptide RRITRASALLDA, Abmart, Shanghai, 1:20 dilution) overnight at 4°C.

Techniques: Transfection, Confocal Microscopy, Staining, Fluorescence, Western Blot

Journal: Frontiers in Immunology

Article Title: Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung

doi: 10.3389/fimmu.2016.00514

Figure Lengend Snippet: Canonical IFNγ signaling is not altered in Socs1 −/− MGL tg mice . (A) Western blot analysis of tyrosine phosphorylated STAT1 (Tyr701). STAT1 and β-actin were used as loading controls. Protein extracts were prepared from BMMs of Socs1 −/− , Socs1 +/− MGL tg , and Socs1 −/− MGL tg mice that were treated with IFNγ (50 ng/ml) as indicated. Quantification using ImageJ is shown in (B) ( n = 4). (C) mRNA expression of interferon target genes iNOS, Irf9 , and Icam-1 are shown normalized to Actin (ActB) expression in BMMs of Socs +/− , Socs1 +/− MGL tg , and Socs1 −/− MGL tg mice treated with IFNγ (50 ng/ml) for 1–6 h ( n = 4). Mean + SD is presented for each group and significance was assessed using two-way ANOVA including Bonferroni post-test.

Article Snippet: Blocking of unspecific binding was performed using 5% BSA solution in 1× TBST [1× TBS, 0.05% (v/v) Tween-20] for at least 1 h. Membranes were stained with antibodies against pY-STAT1 (Tyr701, #9167), STAT1 (#9172), IκBα (#9242), β-Actin (#4970) (Cell Signaling, Leiden, Netherlands; 1:1000), or hybridoma cell supernatant for SOCS1 detection (hybridoma cells newly generated by immunizing mice against the peptide RRITRASALLDA, Abmart, Shanghai, 1:20 dilution) overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Gestational MZD affects phosphorylations of STAT1 and STAT3 in E19 brain total fractions. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. STAT1 and STAT3 contents and phosphorylations in E19 brain total homogenates were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin (top); and band quantifications expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and total STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-actin (top), and band quantifications expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3 and STAT3/β-actin (bottom). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Control, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Gestational MZD affects DNA binding and content of STAT1 and STAT3 in nuclear fractions isolated from E19 brains. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. Nuclear and cytosolic fractions were prepared from E19 brains as described in the Materials and Methods section. (A) EMSA for STAT1 and STAT3 in nuclear and cytosolic fractions. To determine the specificity of each transcription factor-DNA complex, a control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for either the specific (S) or an unspecific (U) transcription factor before the binding assay. Bands were quantified and the ratio nuclear/cytosolic DNA binding (NF/CF) was calculated. (B) Western blots for pY 701 -STAT1, pS 727 -STAT1, and STAT1 (left); and pY 705 -STAT3, pS 727 -STAT3, and STAT3 (right); and hnRNP as the nuclear housekeeping protein. After quantification, results were expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom left), or pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom right). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Binding Assay, Isolation, Control, Incubation, Sequencing, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 total fractions. Total cell fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or zinc -depleted (1.5 μM zinc (1.5Zn)) medium. STAT1 and STAT3 contents and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-tubulin as the housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/β-tubulin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-tubulin (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/β-tubulin (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Phospho-proteomics, Isolation, Incubation, Control, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Zinc deficiency affects nuclear STAT1- and STAT3-DNA binding, and transactivation of STAT1 and STAT3-dependent genes in IMR-32 cells . Nuclear and total cell fractions were isolated after incubating IMR-32 cells for 24 h in control medium (C), or in chelated medium containing 1.5 μM zinc (1.5Zn), or 15 μM zinc (15Zn). (A) Representative image of an EMSA for STAT1 and STAT3 in nuclear fractions (top). A control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for aspecific (S) transcription factor before the binding assay. Bands were quantified, results expressed as the ratio nuclear/total fraction binding (NF/TF) for STAT1 (white bars) and STAT3 (grey bars), and normalized to control values. Results are shown as means±S.E.M. of three independent experiments. (B) Transactivation of STAT1- and STAT3-driven luciferase was measured as described in Materials and Methods in cells incubated for 24 h in control, 1.5Zn or 15Zn medium. Data are expressed as the ratio luciferase activity/β-galactosidase activity. Results are shown as means±S.E.M of three independent experiments. *Significantly different compared to the C groups ( P <0.05, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Binding Assay, Isolation, Control, Incubation, Sequencing, Luciferase, Activity Assay

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 cell nuclear fractions. Nuclear fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or 1.5Zn medium. STAT1 and STAT3 content and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and hnRNP as the nuclear housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and hnRNP (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Phospho-proteomics, Isolation, Incubation, Control, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: α-Lipoic acid prevents zinc deficiency-induced impaired STAT1 and STAT3 nuclear import. Nuclear fraction and cytosolic fraction were isolated from IMR-32 cells incubated for 24 h in control (C), or zinc depleted (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). Western blots for: (A) STAT1 and hnRNP as housekeeping protein in the nuclear fraction (top left), and STAT1 and β-actin as housekeeping protein in the cytosolic fraction (top right), and band quantification expressed as the ratio of total STAT1 nuclear/cytosolic (bottom); (B) STAT3 and hnRNP in the nuclear fraction (top left) and STAT3 and β-actin in the cytosolic fraction (top right), and band quantification expressed as the ratio total STAT3 nuclear/cytosolic fractions (bottom). Results were normalized to controls values, and shown as means±S.E.M. of three independent experiments. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Isolation, Incubation, Control, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: STAT1 and STAT3 require a functional cytoskeleton for nuclear translocation in IMR-32 cells. Nuclear fractions and total fractions were prepared from IMR-32 cells incubated for 24 h in control medium (C) with or without the addition of 0.5 μM vinblastine (Vb), 0.5 μM colchicine (Col), or 0.5 μM cytochalasin D (Cyt D). (A) EMSA for STAT1 in nuclear and total fractions (top), and band quantifications (bottom). (B) EMSA for STAT3 in nuclear and total fractions (top), and band quantifications (bottom). Results were expressed as the ratio nuclear/total fractions of DNA binding (NF/TF) and normalized to their control levels. Results are shown as means±S.E.M. of three independent experiments. *Significantly different compared to C without inhibitors ( P <0.05, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Functional Assay, Translocation Assay, Incubation, Control, Binding Assay

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: α-Lipoic acid differentially affects STAT1 and STAT3 phosphorylation in zinc deficient IMR-32 cells. Total fractions were prepared from IMR-32 cells incubated for 24 h in control (C), or zinc deficient (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). STAT1 and STAT3 content and phosphorylation were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1, and STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, STAT3 and β-actin, and their quantification expressed as pY 705 -STAT3/STAT3, and STAT3/β-actin. Results were normalized to control values and shown as means±S.E.M. of four independent experiments. *, **, *** Significantly different compared to C groups ( P <0.05, P <0.01, and P <0.001, respectively, one-way ANOVA).

Article Snippet: Antibodies for phosphotyrosine-701 STAT1 (pY 701 -STAT1), phosphoserine-727 STAT1 (pS 727 -STAT1), phosphotyrosine-705 STAT3 (pY 705 -STAT3), and phosphoserine-727 STAT3 (pS 727 -STAT3) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Phospho-proteomics, Incubation, Control, Western Blot