Structured Review

Becton Dickinson anti stat1
The effect of TAK1 inhibitor 5Z-7-oxozeaenol on the Nef-induced signaling activation in M2-M Φ . ( a ) M2-M Φ were left untreated (−) or pretreated for 1 h with DMSO or 5Z-7-oxozeaenol at the concentrations indicated ( μ M). Then, the M Φ were left untreated (−) or stimulated with Nef (100 ng/ml) for 30 min and then analyzed by western blotting using the indicated antibodies. ( b ) M2-M Φ were left untreated (−) or pretreated for 1 h with DMSO or 5Z-7-oxozeaenol (0.3 μ M). Then, the M Φ were left untreated or stimulated with Nef (100 ng/ml) for the indicated periods (h) and analyzed by western blotting using the indicated antibodies. ( c ) M2-M Φ were left untreated (−) or preincubated for 1 h with DMSO or 5Z-7-oxozeaenol (0.3 μ M). Then, the M Φ were left untreated or stimulated with granulocyte-macrophages colony-stimulating factor (GM-CSF; 10 ng/ml) for the indicated periods (min) and analyzed by western blotting using the indicated antibodies. <t>p-Stat1:</t> phospho-Stat1, p-Stat3: phospho-Stat3, p-Stat5: phospho-Stat5. ( a – c ) The actin blot is a loading control. Experiments were repeated with M2-M Φ obtained from different donors, and the data shown are representative of three independent experiments with similar results. ERK, extracellular signal–regulated kinase; IKK, inhibitor-κB kinase; JNK, c-Jun N-terminal kinase; Stat, signal transducer and activator of transcription factor
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1) Product Images from "Macropinocytosis and TAK1 mediate anti-inflammatory to pro-inflammatory macrophage differentiation by HIV-1 Nef"

Article Title: Macropinocytosis and TAK1 mediate anti-inflammatory to pro-inflammatory macrophage differentiation by HIV-1 Nef

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.233

The effect of TAK1 inhibitor 5Z-7-oxozeaenol on the Nef-induced signaling activation in M2-M Φ . ( a ) M2-M Φ were left untreated (−) or pretreated for 1 h with DMSO or 5Z-7-oxozeaenol at the concentrations indicated ( μ M). Then, the M Φ were left untreated (−) or stimulated with Nef (100 ng/ml) for 30 min and then analyzed by western blotting using the indicated antibodies. ( b ) M2-M Φ were left untreated (−) or pretreated for 1 h with DMSO or 5Z-7-oxozeaenol (0.3 μ M). Then, the M Φ were left untreated or stimulated with Nef (100 ng/ml) for the indicated periods (h) and analyzed by western blotting using the indicated antibodies. ( c ) M2-M Φ were left untreated (−) or preincubated for 1 h with DMSO or 5Z-7-oxozeaenol (0.3 μ M). Then, the M Φ were left untreated or stimulated with granulocyte-macrophages colony-stimulating factor (GM-CSF; 10 ng/ml) for the indicated periods (min) and analyzed by western blotting using the indicated antibodies. p-Stat1: phospho-Stat1, p-Stat3: phospho-Stat3, p-Stat5: phospho-Stat5. ( a – c ) The actin blot is a loading control. Experiments were repeated with M2-M Φ obtained from different donors, and the data shown are representative of three independent experiments with similar results. ERK, extracellular signal–regulated kinase; IKK, inhibitor-κB kinase; JNK, c-Jun N-terminal kinase; Stat, signal transducer and activator of transcription factor
Figure Legend Snippet: The effect of TAK1 inhibitor 5Z-7-oxozeaenol on the Nef-induced signaling activation in M2-M Φ . ( a ) M2-M Φ were left untreated (−) or pretreated for 1 h with DMSO or 5Z-7-oxozeaenol at the concentrations indicated ( μ M). Then, the M Φ were left untreated (−) or stimulated with Nef (100 ng/ml) for 30 min and then analyzed by western blotting using the indicated antibodies. ( b ) M2-M Φ were left untreated (−) or pretreated for 1 h with DMSO or 5Z-7-oxozeaenol (0.3 μ M). Then, the M Φ were left untreated or stimulated with Nef (100 ng/ml) for the indicated periods (h) and analyzed by western blotting using the indicated antibodies. ( c ) M2-M Φ were left untreated (−) or preincubated for 1 h with DMSO or 5Z-7-oxozeaenol (0.3 μ M). Then, the M Φ were left untreated or stimulated with granulocyte-macrophages colony-stimulating factor (GM-CSF; 10 ng/ml) for the indicated periods (min) and analyzed by western blotting using the indicated antibodies. p-Stat1: phospho-Stat1, p-Stat3: phospho-Stat3, p-Stat5: phospho-Stat5. ( a – c ) The actin blot is a loading control. Experiments were repeated with M2-M Φ obtained from different donors, and the data shown are representative of three independent experiments with similar results. ERK, extracellular signal–regulated kinase; IKK, inhibitor-κB kinase; JNK, c-Jun N-terminal kinase; Stat, signal transducer and activator of transcription factor

Techniques Used: Activation Assay, Western Blot

2) Product Images from "Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells"

Article Title: Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells

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

doi: 10.1073/pnas.0804231105

Ahr regulates the activation of Stat1 in Th17 cell development. ( A ) MACS-sorted naïve T cells were cultured with anti-CD3/CD28 beads and stimulated with IL-6 or TGF-β, either alone or combined, for 2 days. Whole cell lysates were immunoprecipitated with anti-Ahr antibody, after which Stat1, Stat3, Stat5, Stat6, and Ahr were detected with Western blotting. IP, immunoprecipitation; IB, immunoblot. ( B ) Naïve T cells isolated from Ahr WT and He mice were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6 in the presence or absence of IFN-γ for 3 days, followed by re-stimulation with PMA and ionomycin for 5 h and with GolgiStop (final 2 h), and then staining for intracellular cytokines. Dot plots show intracellular staining for IFN-γ and IL-17. ( C and D ) Naïve T cells isolated from Ahr WT and KO splenocytes were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6 for 30 min or 24 h, fixed and permeabilized in 90% methanol, and finally stained with Alexa Fluor 488-conjugated phospho-Stat1 and PE-conjugated phospho-Stat3. Intracellular levels of phospho-Stat1 ( C ) and Stat3 ( D ) were measured by means of flow cytometry. These results are representative of three independent experiments.
Figure Legend Snippet: Ahr regulates the activation of Stat1 in Th17 cell development. ( A ) MACS-sorted naïve T cells were cultured with anti-CD3/CD28 beads and stimulated with IL-6 or TGF-β, either alone or combined, for 2 days. Whole cell lysates were immunoprecipitated with anti-Ahr antibody, after which Stat1, Stat3, Stat5, Stat6, and Ahr were detected with Western blotting. IP, immunoprecipitation; IB, immunoblot. ( B ) Naïve T cells isolated from Ahr WT and He mice were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6 in the presence or absence of IFN-γ for 3 days, followed by re-stimulation with PMA and ionomycin for 5 h and with GolgiStop (final 2 h), and then staining for intracellular cytokines. Dot plots show intracellular staining for IFN-γ and IL-17. ( C and D ) Naïve T cells isolated from Ahr WT and KO splenocytes were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6 for 30 min or 24 h, fixed and permeabilized in 90% methanol, and finally stained with Alexa Fluor 488-conjugated phospho-Stat1 and PE-conjugated phospho-Stat3. Intracellular levels of phospho-Stat1 ( C ) and Stat3 ( D ) were measured by means of flow cytometry. These results are representative of three independent experiments.

Techniques Used: Activation Assay, Magnetic Cell Separation, Cell Culture, Immunoprecipitation, Western Blot, Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry

3) Product Images from "SETD1B activates iNOS expression in myeloid-derived suppressor cells"

Article Title: SETD1B activates iNOS expression in myeloid-derived suppressor cells

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-16-2238

The IFNγ and NF-κB signaling pathways and MDSCs A . MDSCs were purified from the spleens of 4T1 tumor-bearing (TB, n=3) and their equivalent in tumor-free (TF, n=3) mice and analyzed by Western blotting for STAT1, pSTAT1, p65, p50, RelB, c-Rel, and p100/52. β-actin was used as a normalization control. B . 4T1 conditioned media was collected from 4T1 tumor cell culture flasks. BM cells from three WT mice were cultured in the presence of 4T1 conditioned media for 6 days. Cells were stained with IgG- or CD11b- and Gr1-specific mAb and analyzed by flow cytometry. Shown is the phenotype of the 4T1 conditioned media-induced MDSCs. C . CD3 + T cells were purified from the spleen of a tumor-free WT mouse and labeled with CFSE. The labeled T cells were then cultured in the absence or presence of 4T1 conditioned media-induced MDSCs at a 2:1 ratio for 3 days and analyzed for CFSE intensity by flow cytometry. Shown are representative data of proliferation of T cells from one of three replicates. D . BM cells from three WT mice were cultured in the presence of 4T1 conditioned media for 6 days. The resultant MDSCs were then either untreated (control) or treated with IFNγ (100 U/ml) or TNFα (100 U/ml) for approximately 20 hours, respectively. Cells were then analyzed by Western blotting for the indicated proteins. β-actin was used as a normalization control. E . BM cells from WT (n=3) and IRF8 KO (n=3) mice were cultured in the presence of 4T1 conditioned media for 6 days and then treated with IFNγ (100 U/ml) for approximately 20 hours. Cells were then analyzed by qPCR for iNOS mRNA level.
Figure Legend Snippet: The IFNγ and NF-κB signaling pathways and MDSCs A . MDSCs were purified from the spleens of 4T1 tumor-bearing (TB, n=3) and their equivalent in tumor-free (TF, n=3) mice and analyzed by Western blotting for STAT1, pSTAT1, p65, p50, RelB, c-Rel, and p100/52. β-actin was used as a normalization control. B . 4T1 conditioned media was collected from 4T1 tumor cell culture flasks. BM cells from three WT mice were cultured in the presence of 4T1 conditioned media for 6 days. Cells were stained with IgG- or CD11b- and Gr1-specific mAb and analyzed by flow cytometry. Shown is the phenotype of the 4T1 conditioned media-induced MDSCs. C . CD3 + T cells were purified from the spleen of a tumor-free WT mouse and labeled with CFSE. The labeled T cells were then cultured in the absence or presence of 4T1 conditioned media-induced MDSCs at a 2:1 ratio for 3 days and analyzed for CFSE intensity by flow cytometry. Shown are representative data of proliferation of T cells from one of three replicates. D . BM cells from three WT mice were cultured in the presence of 4T1 conditioned media for 6 days. The resultant MDSCs were then either untreated (control) or treated with IFNγ (100 U/ml) or TNFα (100 U/ml) for approximately 20 hours, respectively. Cells were then analyzed by Western blotting for the indicated proteins. β-actin was used as a normalization control. E . BM cells from WT (n=3) and IRF8 KO (n=3) mice were cultured in the presence of 4T1 conditioned media for 6 days and then treated with IFNγ (100 U/ml) for approximately 20 hours. Cells were then analyzed by qPCR for iNOS mRNA level.

Techniques Used: Purification, Mouse Assay, Western Blot, Cell Culture, Staining, Flow Cytometry, Cytometry, Labeling, Real-time Polymerase Chain Reaction

4) Product Images from "Single Cell Based Phosphorylation Profiling Identifies Alterations in Toll-Like Receptor 7 and 9 Signaling in Patients With Primary Sjögren's Syndrome"

Article Title: Single Cell Based Phosphorylation Profiling Identifies Alterations in Toll-Like Receptor 7 and 9 Signaling in Patients With Primary Sjögren's Syndrome

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00281

TLR stimulation results in different phosphorylation profiles in B cells, T cells, and NK cells of pSS patients compared to healthy controls. Phosphorylation levels of NF-κB, P38, ERK1/2, STAT4 Y693, STAT5 Y694, STAT1 Y701, STAT1 S727, STAT3 Y705, and STAT3 S727 were analyzed by flow cytometry at different time points after stimulation with TLR7 and −9 ligands. Comparisons of change of phosphorylation levels (ΔMdFI) between pSS patient (black) and healthy donors (blue) are given. Comparisons between pairs were done using an Unpaired Mann-Whitney test. Line graphs show the median and 25–75 percentiles. Differences were considered statistically significant when p ≤ 0.05, with significance indicated as * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001 and **** ≤ 0.0001. The data represents 25 healthy controls and 25 patients pooled from 13 independent experiments.
Figure Legend Snippet: TLR stimulation results in different phosphorylation profiles in B cells, T cells, and NK cells of pSS patients compared to healthy controls. Phosphorylation levels of NF-κB, P38, ERK1/2, STAT4 Y693, STAT5 Y694, STAT1 Y701, STAT1 S727, STAT3 Y705, and STAT3 S727 were analyzed by flow cytometry at different time points after stimulation with TLR7 and −9 ligands. Comparisons of change of phosphorylation levels (ΔMdFI) between pSS patient (black) and healthy donors (blue) are given. Comparisons between pairs were done using an Unpaired Mann-Whitney test. Line graphs show the median and 25–75 percentiles. Differences were considered statistically significant when p ≤ 0.05, with significance indicated as * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001 and **** ≤ 0.0001. The data represents 25 healthy controls and 25 patients pooled from 13 independent experiments.

Techniques Used: Flow Cytometry, Cytometry, MANN-WHITNEY

Basal phosphorylation profiles in B cells, T cells and NK cells of pSS patients differ compared to healthy controls. Basal phosphorylation levels of NF-κB, P38, ERK1/2, STAT4 Y693, STAT5 Y694, STAT1 Y701, STAT1 S727, STAT3 Y705, and STAT3 S727 were analyzed by flow cytometry in T cells, B cells and NK cells are given in (A) . Comparisons of phosphorylation levels (MdFI) between healthy donor (blue) and pSS patient (black). Comparisons between pairs were done using an Unpaired Mann-Whitney test. Graphs show the median, 25–75 percentiles and minimum and maximum. Differences were considered statistically significant when p ≤ 0.05, with significance indicated as * ≤ 0.05, ** ≤ 0.01, and **** ≤ 0.0001. PCA analysis of the profiles is given in (B–F) , disease status is highlighted in (C) , SSA autoantibody positivity in (D) , presence of EGM in (E) , and medication (DMARDs or corticosteroids) use in (F) , with healthy donors, blue squares; pSS patients, black circles; SSA+, EGM+, or medicated patients, red diamonds ( D–F , respectively). The loading plot, which contains information about the variable for the corresponding PCA is given in (B) , with variable indicated by vectors and the key to the right. Variables contributing little to the PCA are plotted around the center as denoted by the gray axis, while variables that have high contributions are plotted further from the axes. After initial calculation of principal components, the model was recalculated with only variable explaining > 50% of the variance retained. The data represents 25 healthy controls and 25 patients pooled from 13 independent experiments.
Figure Legend Snippet: Basal phosphorylation profiles in B cells, T cells and NK cells of pSS patients differ compared to healthy controls. Basal phosphorylation levels of NF-κB, P38, ERK1/2, STAT4 Y693, STAT5 Y694, STAT1 Y701, STAT1 S727, STAT3 Y705, and STAT3 S727 were analyzed by flow cytometry in T cells, B cells and NK cells are given in (A) . Comparisons of phosphorylation levels (MdFI) between healthy donor (blue) and pSS patient (black). Comparisons between pairs were done using an Unpaired Mann-Whitney test. Graphs show the median, 25–75 percentiles and minimum and maximum. Differences were considered statistically significant when p ≤ 0.05, with significance indicated as * ≤ 0.05, ** ≤ 0.01, and **** ≤ 0.0001. PCA analysis of the profiles is given in (B–F) , disease status is highlighted in (C) , SSA autoantibody positivity in (D) , presence of EGM in (E) , and medication (DMARDs or corticosteroids) use in (F) , with healthy donors, blue squares; pSS patients, black circles; SSA+, EGM+, or medicated patients, red diamonds ( D–F , respectively). The loading plot, which contains information about the variable for the corresponding PCA is given in (B) , with variable indicated by vectors and the key to the right. Variables contributing little to the PCA are plotted around the center as denoted by the gray axis, while variables that have high contributions are plotted further from the axes. After initial calculation of principal components, the model was recalculated with only variable explaining > 50% of the variance retained. The data represents 25 healthy controls and 25 patients pooled from 13 independent experiments.

Techniques Used: Flow Cytometry, Cytometry, MANN-WHITNEY

5) Product Images from "Mediators of the JAK/STAT Signaling Pathway in Human Spermatozoa 1"

Article Title: Mediators of the JAK/STAT Signaling Pathway in Human Spermatozoa 1

Journal: Biology of Reproduction

doi: 10.1095/biolreprod.111.092379

STAT proteins in sperm subcellular fractions. A ) Following fractionation by nitrogen cavitation, sonication, and different centrifugation, sperm proteins were submitted to electrophoresis and transferred on PVDF membrane for immunoblotting with anti-STAT1,
Figure Legend Snippet: STAT proteins in sperm subcellular fractions. A ) Following fractionation by nitrogen cavitation, sonication, and different centrifugation, sperm proteins were submitted to electrophoresis and transferred on PVDF membrane for immunoblotting with anti-STAT1,

Techniques Used: Fractionation, Sonication, Centrifugation, Electrophoresis

Localization of IL6R, IL6ST, JAK1, STAT1, and STAT4 on sperm. Fixed and permeabilized spermatozoa were submitted to indirect immunofluorescence using an anti-JAK1 or anti-STAT1 monoclonal antibody ( D and E , respectively) or an anti-IL6R, anti-IL6ST, or
Figure Legend Snippet: Localization of IL6R, IL6ST, JAK1, STAT1, and STAT4 on sperm. Fixed and permeabilized spermatozoa were submitted to indirect immunofluorescence using an anti-JAK1 or anti-STAT1 monoclonal antibody ( D and E , respectively) or an anti-IL6R, anti-IL6ST, or

Techniques Used: Immunofluorescence

6) Product Images from "PIM-2 protein kinase negatively regulates T cell responses in transplantation and tumor immunity"

Article Title: PIM-2 protein kinase negatively regulates T cell responses in transplantation and tumor immunity

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI95407

PIM-2–deficient T cells augment antitumor activity via IL-9R/STAT1 pathway. ( A ) Bar graphs show fold change of mRNA expression of HIF1α , IL-9R , GZMB , IL-10 , SOCS-1 , and TCF7 on TILs evaluated by quantitative PCR ( n = 4 mice per group). ( B ) Histogram analyses show IL-9R expression from TILs, and quantified MFI is shown in the bar graph. ( C ) Phospho-STAT1 was measured on T cells isolated from tumor sites and restimulated with IL-9 (20 ng/ml) in vitro. Percentages of p-STAT1 are shown in the bar graph ( n = 3 mice per group). Data represent mean ± SEM by 2-tailed Student’s t test. * P
Figure Legend Snippet: PIM-2–deficient T cells augment antitumor activity via IL-9R/STAT1 pathway. ( A ) Bar graphs show fold change of mRNA expression of HIF1α , IL-9R , GZMB , IL-10 , SOCS-1 , and TCF7 on TILs evaluated by quantitative PCR ( n = 4 mice per group). ( B ) Histogram analyses show IL-9R expression from TILs, and quantified MFI is shown in the bar graph. ( C ) Phospho-STAT1 was measured on T cells isolated from tumor sites and restimulated with IL-9 (20 ng/ml) in vitro. Percentages of p-STAT1 are shown in the bar graph ( n = 3 mice per group). Data represent mean ± SEM by 2-tailed Student’s t test. * P

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

Inhibition of PIM-2 kinase in T cells induces severe GVHD while mediating cytolytic function against the tumor. The absence of PIM-2 isoform after allostimulation increases T cell proliferation, proinflammatory cytokines, and CD4 + T cell differentiation into a Th1 subset and causes tissue damage in a GVHD model. Despite the increased inflammatory responses, PIM-2–deficient T cells upregulate IL-9R, STAT1, and FasL expression and augment antitumor activity. APC, antigen-presenting cell.
Figure Legend Snippet: Inhibition of PIM-2 kinase in T cells induces severe GVHD while mediating cytolytic function against the tumor. The absence of PIM-2 isoform after allostimulation increases T cell proliferation, proinflammatory cytokines, and CD4 + T cell differentiation into a Th1 subset and causes tissue damage in a GVHD model. Despite the increased inflammatory responses, PIM-2–deficient T cells upregulate IL-9R, STAT1, and FasL expression and augment antitumor activity. APC, antigen-presenting cell.

Techniques Used: Inhibition, Cell Differentiation, Expressing, Activity Assay

7) Product Images from "Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis"

Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis

Journal: Cell reports

doi: 10.1016/j.celrep.2018.11.050

IL-10 Induces the Activation of STAT3 that Binds to the dnmt1 and dnmt3b Promoters in Colon Epithelial and Carcinoma Cells (A) WT C57BL/6 mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected from mice at the indicated time points and analyzed by western blotting for STAT1 and STAT3 protein levels. β-Actin was used as a normalization control. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 2 hr and analyzed for the indicated proteins by western blotting. (C) Top: structure of the Dnmt1 promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt1 transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using immunoglobulin G (IgG) control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt1 promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (D) Top: structure of the Dnmt3b promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt3b transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using IgG control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt3b promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (E and F) The human DNMT1 (E) and DNMT3b (F) promoter DNA fragments were amplified by PCR from the two indicated regions (top: P1 and P2 for DNMT1 , and P3 and P4 for DNMT3b ) and cloned to the pGL3 vector. pGL3 vectors containing the P1, P2, P3, or P4 DNA fragments were transiently transfected to CCD841 and HT29 cells, respectively, overnight. Cells were either untreated (control) or treated with IL-10 (100 ng/mL) for 4 hr. Cells were lysated and analyzed for luciferase activity. Bar, SD.
Figure Legend Snippet: IL-10 Induces the Activation of STAT3 that Binds to the dnmt1 and dnmt3b Promoters in Colon Epithelial and Carcinoma Cells (A) WT C57BL/6 mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected from mice at the indicated time points and analyzed by western blotting for STAT1 and STAT3 protein levels. β-Actin was used as a normalization control. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 2 hr and analyzed for the indicated proteins by western blotting. (C) Top: structure of the Dnmt1 promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt1 transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using immunoglobulin G (IgG) control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt1 promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (D) Top: structure of the Dnmt3b promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt3b transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using IgG control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt3b promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (E and F) The human DNMT1 (E) and DNMT3b (F) promoter DNA fragments were amplified by PCR from the two indicated regions (top: P1 and P2 for DNMT1 , and P3 and P4 for DNMT3b ) and cloned to the pGL3 vector. pGL3 vectors containing the P1, P2, P3, or P4 DNA fragments were transiently transfected to CCD841 and HT29 cells, respectively, overnight. Cells were either untreated (control) or treated with IL-10 (100 ng/mL) for 4 hr. Cells were lysated and analyzed for luciferase activity. Bar, SD.

Techniques Used: Activation Assay, Mouse Assay, Western Blot, Recombinant, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Activity Assay

8) Product Images from "Tat engagement of p38 MAP kinase and IRF7 pathways leads to activation of interferon-stimulated genes in antigen-presenting cells"

Article Title: Tat engagement of p38 MAP kinase and IRF7 pathways leads to activation of interferon-stimulated genes in antigen-presenting cells

Journal: Blood

doi: 10.1182/blood-2012-10-461566

MAP2K6 and IRF7 induce Tat-mediated cellular gene modulation via the p38-STAT1 pathway. mRNA levels of ISGs in KG-iDC (A) and type I IFNR2-deficient 5UA cells (B) transfected with MAP2K6 and IRF7 siRNAs and infected with Ad-Tat SF2 /Ad-tTA or Ad-tTA alone.
Figure Legend Snippet: MAP2K6 and IRF7 induce Tat-mediated cellular gene modulation via the p38-STAT1 pathway. mRNA levels of ISGs in KG-iDC (A) and type I IFNR2-deficient 5UA cells (B) transfected with MAP2K6 and IRF7 siRNAs and infected with Ad-Tat SF2 /Ad-tTA or Ad-tTA alone.

Techniques Used: Transfection, Infection

Schematic of the pathway triggered by Tat and leading to activation of ISGs by Tat in APCs infected by HIV. (A) Tat triggers signaling pathways that activate ISGs by associating with MAP2K3 and MAP2K6 kinases, which in turn activate p38 MAPK and STAT1,
Figure Legend Snippet: Schematic of the pathway triggered by Tat and leading to activation of ISGs by Tat in APCs infected by HIV. (A) Tat triggers signaling pathways that activate ISGs by associating with MAP2K3 and MAP2K6 kinases, which in turn activate p38 MAPK and STAT1,

Techniques Used: Activation Assay, Infection

9) Product Images from "Anti-Inflammatory Effect of 3-Bromo-4,5-Dihydroxybenzaldehyde, a Component of Polysiphonia morrowii, In Vivo and In Vitro"

Article Title: Anti-Inflammatory Effect of 3-Bromo-4,5-Dihydroxybenzaldehyde, a Component of Polysiphonia morrowii, In Vivo and In Vitro

Journal: Toxicological Research

doi: 10.5487/TR.2017.33.4.325

Effects of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) on the phosphorylation of STAT1 at Tyr701 and Ser727 residues in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. RAW 264.7 cells (5.0 × 10 5 cells/mL) were pre-incubated for 18 hr. Cells were pretreated with BDB (12.5 to 100 μM) for 120min and stimulated with LPS (1 μg/mL) for 120min. Expression of phosphorylated STAT1 at Tyr701 and Ser727, STAT1, SOCS1, and β-actin was determined by western blotting of whole cell lysates with the indicated antibodies.
Figure Legend Snippet: Effects of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) on the phosphorylation of STAT1 at Tyr701 and Ser727 residues in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. RAW 264.7 cells (5.0 × 10 5 cells/mL) were pre-incubated for 18 hr. Cells were pretreated with BDB (12.5 to 100 μM) for 120min and stimulated with LPS (1 μg/mL) for 120min. Expression of phosphorylated STAT1 at Tyr701 and Ser727, STAT1, SOCS1, and β-actin was determined by western blotting of whole cell lysates with the indicated antibodies.

Techniques Used: Incubation, Expressing, Western Blot

10) Product Images from "The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth"

Article Title: The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth

Journal: Oncotarget

doi: 10.18632/oncotarget.9315

Immunohistochemical analyses of xenografted tumors of CRC cell lines with STAT3 knockdown H E and immunohistochemical staining of xenografts for STAT3, STAT1 and cleaved CASPASE 3 for the respective cell lines – a. HCT116, b. SW620, c. HT29 and d. LS174T. Quantification of the cleaved CASPASE 3 (right panel) was done using HistoQuest TM software, from three images taken from three independent tumors. Mean values are shown, error bars are SEM and * p
Figure Legend Snippet: Immunohistochemical analyses of xenografted tumors of CRC cell lines with STAT3 knockdown H E and immunohistochemical staining of xenografts for STAT3, STAT1 and cleaved CASPASE 3 for the respective cell lines – a. HCT116, b. SW620, c. HT29 and d. LS174T. Quantification of the cleaved CASPASE 3 (right panel) was done using HistoQuest TM software, from three images taken from three independent tumors. Mean values are shown, error bars are SEM and * p

Techniques Used: Immunohistochemistry, Staining, Software

IL-6-dependent activation and subcellular localization of STAT3 and STAT1 in CRC cell lines a. Western blot analysis of extracts from CRC cell lines stimulated with 10 ng/ml of IL-6, for 20 min. b. DNA binding assay (EMSA) for STAT1/3 binding on the SIEm67 STAT1/3 response element, with CRC extracts stimulated with or without IL-6; including pre-incubation with STAT1 and/or STAT3 specific antibodies to analyse ‘supershift’ of the complexes. c. Immunoprecipitation of STAT3 from CRC cell lines followed by Western blot analysis with anti-STAT1 and STAT3 antibodies. d. Immunofluorescence and confocal microscopy for detection of intracellular localization of STAT1. e. Quantification of percentage of nuclear STAT1 is presented as mean values (± SEM). f. Immunofluorescence and confocal microscopy for detection of pYSTAT3 upon IL-6 stimulation.
Figure Legend Snippet: IL-6-dependent activation and subcellular localization of STAT3 and STAT1 in CRC cell lines a. Western blot analysis of extracts from CRC cell lines stimulated with 10 ng/ml of IL-6, for 20 min. b. DNA binding assay (EMSA) for STAT1/3 binding on the SIEm67 STAT1/3 response element, with CRC extracts stimulated with or without IL-6; including pre-incubation with STAT1 and/or STAT3 specific antibodies to analyse ‘supershift’ of the complexes. c. Immunoprecipitation of STAT3 from CRC cell lines followed by Western blot analysis with anti-STAT1 and STAT3 antibodies. d. Immunofluorescence and confocal microscopy for detection of intracellular localization of STAT1. e. Quantification of percentage of nuclear STAT1 is presented as mean values (± SEM). f. Immunofluorescence and confocal microscopy for detection of pYSTAT3 upon IL-6 stimulation.

Techniques Used: Activation Assay, Western Blot, DNA Binding Assay, Binding Assay, Incubation, Immunoprecipitation, Immunofluorescence, Confocal Microscopy

Differential effect of STAT3 knockdown on STAT1 expression in CRC cell lines a. Real time PCR analysis for quantification of STAT3 mRNA levels upon shRNA mediated knockdown. Mean values are shown, error bars are SEM and * p
Figure Legend Snippet: Differential effect of STAT3 knockdown on STAT1 expression in CRC cell lines a. Real time PCR analysis for quantification of STAT3 mRNA levels upon shRNA mediated knockdown. Mean values are shown, error bars are SEM and * p

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

Correlation of combined STAT1 and IL-6Rα expression in CRC tissue with patient survival Examples of patient TMAs with IL-6Rα staining, showing different levels of expression. a. Immunohistochemical staining was scored as negative (score 0), weak (score 1), moderate (score 2) or strong (score 3). b. Patient survival upon concomitant presence of IL-6Rα and nuclear STAT3 and c. concomitant absence of IL-6Rα and cytoplasmic STAT1.
Figure Legend Snippet: Correlation of combined STAT1 and IL-6Rα expression in CRC tissue with patient survival Examples of patient TMAs with IL-6Rα staining, showing different levels of expression. a. Immunohistochemical staining was scored as negative (score 0), weak (score 1), moderate (score 2) or strong (score 3). b. Patient survival upon concomitant presence of IL-6Rα and nuclear STAT3 and c. concomitant absence of IL-6Rα and cytoplasmic STAT1.

Techniques Used: Expressing, Staining, Immunohistochemistry

Lower median survival in patients with concomitant absence of nuclear STAT1 and STAT3 Examples of patient TMAs with nuclear STAT1 and STAT3 staining, showing different levels of STAT expression. a. Immunohistochemical staining was scored by two board certified pathologists as negative (score 0), weak (score 1), moderate (score 2) or strong (score 3). Patients show reduced survival upon concomitant absence of b. nuclear and c. cytoplasmic STAT1 and STAT3. d. Higher nuclear STAT1/STAT3 ratio correlates with increased patient survival.
Figure Legend Snippet: Lower median survival in patients with concomitant absence of nuclear STAT1 and STAT3 Examples of patient TMAs with nuclear STAT1 and STAT3 staining, showing different levels of STAT expression. a. Immunohistochemical staining was scored by two board certified pathologists as negative (score 0), weak (score 1), moderate (score 2) or strong (score 3). Patients show reduced survival upon concomitant absence of b. nuclear and c. cytoplasmic STAT1 and STAT3. d. Higher nuclear STAT1/STAT3 ratio correlates with increased patient survival.

Techniques Used: Staining, Expressing, Immunohistochemistry

11) Product Images from "Anti-Inflammatory Effect of 3-Bromo-4,5-Dihydroxybenzaldehyde, a Component of Polysiphonia morrowii, In Vivo and In Vitro"

Article Title: Anti-Inflammatory Effect of 3-Bromo-4,5-Dihydroxybenzaldehyde, a Component of Polysiphonia morrowii, In Vivo and In Vitro

Journal: Toxicological Research

doi: 10.5487/TR.2017.33.4.325

Effects of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) on the phosphorylation of STAT1 at Tyr701 and Ser727 residues in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. RAW 264.7 cells (5.0 × 10 5 cells/mL) were pre-incubated for 18 hr. Cells
Figure Legend Snippet: Effects of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) on the phosphorylation of STAT1 at Tyr701 and Ser727 residues in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. RAW 264.7 cells (5.0 × 10 5 cells/mL) were pre-incubated for 18 hr. Cells

Techniques Used: Incubation

12) Product Images from "The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells"

Article Title: The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells

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

doi: 10.4049/jimmunol.1400084

Phosphorylation of STAT1 and STAT5 is Confirmed by Flow Cytometry and Immunoblotting. Freshly isolated PBMCs were treated with multiple concentrations of sorafenib for 30 minutes and then stimulated with 8 nM IL-2, 10 5 U/mL IFN-α, 10 ng/mL IFN-γ,
Figure Legend Snippet: Phosphorylation of STAT1 and STAT5 is Confirmed by Flow Cytometry and Immunoblotting. Freshly isolated PBMCs were treated with multiple concentrations of sorafenib for 30 minutes and then stimulated with 8 nM IL-2, 10 5 U/mL IFN-α, 10 ng/mL IFN-γ,

Techniques Used: Flow Cytometry, Cytometry, Isolation

13) Product Images from "Intracellular Delivery of a Cell-Penetrating SOCS1 that Targets IFN-? Signaling"

Article Title: Intracellular Delivery of a Cell-Penetrating SOCS1 that Targets IFN-? Signaling

Journal: Science signaling

doi: 10.1126/scisignal.1162191

CP-SOCS1 inhibits IFN-γ– induced phosphorylation of STAT1 in AMJ2.C8 cells and BMDMs. LPS-hyporesponsive AMJ2.C8 cells ( A and B ) and BMDMs ( C and D ) were treated with diluent or the indicated concentrations of non–CP-SOCS1 or CP-SOCS1
Figure Legend Snippet: CP-SOCS1 inhibits IFN-γ– induced phosphorylation of STAT1 in AMJ2.C8 cells and BMDMs. LPS-hyporesponsive AMJ2.C8 cells ( A and B ) and BMDMs ( C and D ) were treated with diluent or the indicated concentrations of non–CP-SOCS1 or CP-SOCS1

Techniques Used:

14) Product Images from "Data on quantification of signaling pathways activated by KIT and PDGFRA mutants"

Article Title: Data on quantification of signaling pathways activated by KIT and PDGFRA mutants

Journal: Data in Brief

doi: 10.1016/j.dib.2016.10.026

Activation of STAT species by GIST mutants. (a) Nuclear translocation of STAT species. Nuclear extracts were prepared as previously done [4] , diluted in 4 times Laemmli buffer and subjected to Western blot analysis. Phosphorylation status of STAT5, STAT3 and STAT1 is shown for nuclear extract prepared from Hek293 cells expressing KIT WT, KIT Ex11, KIT Ex9 and KIT V559D. (b) Induction of known STAT target genes by GIST mutants. Gene expression level of known STAT target genes, previously identified to be induced in PDGFRA GIST mutants [3] , was retrieved from micro-array data and presented as heat map. Grey boxes indicate that the genes are not part of the DEG list for the corresponding mutant (FDR > 0.05 or AbsFC
Figure Legend Snippet: Activation of STAT species by GIST mutants. (a) Nuclear translocation of STAT species. Nuclear extracts were prepared as previously done [4] , diluted in 4 times Laemmli buffer and subjected to Western blot analysis. Phosphorylation status of STAT5, STAT3 and STAT1 is shown for nuclear extract prepared from Hek293 cells expressing KIT WT, KIT Ex11, KIT Ex9 and KIT V559D. (b) Induction of known STAT target genes by GIST mutants. Gene expression level of known STAT target genes, previously identified to be induced in PDGFRA GIST mutants [3] , was retrieved from micro-array data and presented as heat map. Grey boxes indicate that the genes are not part of the DEG list for the corresponding mutant (FDR > 0.05 or AbsFC

Techniques Used: Activation Assay, Translocation Assay, Western Blot, Expressing, Microarray, Mutagenesis

15) Product Images from "A Novel Role for IFN-Stimulated Gene Factor 3II in IFN-? Signaling and Induction of Antiviral Activity in Human Cells"

Article Title: A Novel Role for IFN-Stimulated Gene Factor 3II in IFN-? Signaling and Induction of Antiviral Activity in Human Cells

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

doi: 10.4049/jimmunol.1001359

Neutralization of STAT1 activation in IFN-γ–treated cells and accumulation of ISGF3 lacking STAT2 phosphorylation. A , STAT2 Abs were used to coimmunoprecipitate the ISGF3 complex in A549 cells. Cells were left untreated ( lane 6 ) or treated
Figure Legend Snippet: Neutralization of STAT1 activation in IFN-γ–treated cells and accumulation of ISGF3 lacking STAT2 phosphorylation. A , STAT2 Abs were used to coimmunoprecipitate the ISGF3 complex in A549 cells. Cells were left untreated ( lane 6 ) or treated

Techniques Used: Neutralization, Activation Assay

Gene expression of select ISGs in IFN-γ–treated cells after neutralization with IFNGR1 Ab. A , IFN-treated cells were examined by Western blot for phosphorylation of STAT1 (at Y701) as an indicator of autocrine or paracrine IFN action.
Figure Legend Snippet: Gene expression of select ISGs in IFN-γ–treated cells after neutralization with IFNGR1 Ab. A , IFN-treated cells were examined by Western blot for phosphorylation of STAT1 (at Y701) as an indicator of autocrine or paracrine IFN action.

Techniques Used: Expressing, Neutralization, Western Blot

STAT1 signaling response in A549 cells treated with IFN-α2a and IFN-γ. Western blot of A549 cells treated with IFN-α2a and IFN-γ showing kinetics of STAT1 (via phosphorylation at Y701) and STAT2 (via phosphorylation at
Figure Legend Snippet: STAT1 signaling response in A549 cells treated with IFN-α2a and IFN-γ. Western blot of A549 cells treated with IFN-α2a and IFN-γ showing kinetics of STAT1 (via phosphorylation at Y701) and STAT2 (via phosphorylation at

Techniques Used: Western Blot

Occupancy of the PKR promoter by ( A ) STAT1, ( B ) STAT2, and ( C ) IRF9 after IFN-γ and IFN-α2a treatment. Cross-linked, sheared chromatin ~1 kb in length from cells left untreated or treated with 10 IU/ml IFN-γ or 10 IU/ml IFN-α2a
Figure Legend Snippet: Occupancy of the PKR promoter by ( A ) STAT1, ( B ) STAT2, and ( C ) IRF9 after IFN-γ and IFN-α2a treatment. Cross-linked, sheared chromatin ~1 kb in length from cells left untreated or treated with 10 IU/ml IFN-γ or 10 IU/ml IFN-α2a

Techniques Used:

16) Product Images from "Ebola virus VP24 targets a unique NLS-binding site on karyopherin5 to selectively compete with nuclear import of phosphorylated STAT1"

Article Title: Ebola virus VP24 targets a unique NLS-binding site on karyopherin5 to selectively compete with nuclear import of phosphorylated STAT1

Journal: Cell host & microbe

doi: 10.1016/j.chom.2014.07.008

Recognition of a shared ncNLS binding site on KPNA by eVP24 is important for inhibiting PY-STAT1 nuclear localization and ISG induction (A-B) eVP24/KPNA5 interface mutants R137A and cluster 3 mut fail to inhibit IFN-mediated STAT1 nuclear translocation. Vero cells were mock-untreated or treated with IFN-β for 30 minutes to induce STAT1 nuclear localization ((E) vector + IFN-β). Ectopic expression of eVP24 WT or mutants differentially affects PY-STAT1 translocation to the nucleus after IFN-β treatment. White arrows highlight IFN-β treated cells that also express eVP24 WT or eVP24 mutants. Representative data from one of two independent experiments is shown. ( C ) The ability of eVP24 to inhibit induction of ISG54 was assessed. 293T cells were co-transfected with an ISG54 firefly luciferase reporter, a constitutively expressed Renilla luciferase plasmid and the indicated eVP24 expression plasmids. The values represent the mean and SEM of six samples, and statistical significance was assessed by a one-way ANOVA comparing individual mutants to the corresponding eVP24 wt transfection, where ***p
Figure Legend Snippet: Recognition of a shared ncNLS binding site on KPNA by eVP24 is important for inhibiting PY-STAT1 nuclear localization and ISG induction (A-B) eVP24/KPNA5 interface mutants R137A and cluster 3 mut fail to inhibit IFN-mediated STAT1 nuclear translocation. Vero cells were mock-untreated or treated with IFN-β for 30 minutes to induce STAT1 nuclear localization ((E) vector + IFN-β). Ectopic expression of eVP24 WT or mutants differentially affects PY-STAT1 translocation to the nucleus after IFN-β treatment. White arrows highlight IFN-β treated cells that also express eVP24 WT or eVP24 mutants. Representative data from one of two independent experiments is shown. ( C ) The ability of eVP24 to inhibit induction of ISG54 was assessed. 293T cells were co-transfected with an ISG54 firefly luciferase reporter, a constitutively expressed Renilla luciferase plasmid and the indicated eVP24 expression plasmids. The values represent the mean and SEM of six samples, and statistical significance was assessed by a one-way ANOVA comparing individual mutants to the corresponding eVP24 wt transfection, where ***p

Techniques Used: Binding Assay, Translocation Assay, Plasmid Preparation, Expressing, Transfection, Luciferase

eVP24 and PY-STAT1 share an overlapping binding site on KPNA5 293T cells were treated with human IFNβ (1000U/mL) for 30 minutes. Co-IPs with HA or Flag antibody were performed as indicated on the 293T cell lysates transfected with (A) HA-tagged eVP24 or Nipah virus V protein (NipV), (B) KPNA5 truncation mutants and (C-D) .
Figure Legend Snippet: eVP24 and PY-STAT1 share an overlapping binding site on KPNA5 293T cells were treated with human IFNβ (1000U/mL) for 30 minutes. Co-IPs with HA or Flag antibody were performed as indicated on the 293T cell lysates transfected with (A) HA-tagged eVP24 or Nipah virus V protein (NipV), (B) KPNA5 truncation mutants and (C-D) .

Techniques Used: Binding Assay, Transfection

17) Product Images from "IFN-? Upregulates Survivin and Ifi202 Expression to Induce Survival and Proliferation of Tumor-Specific T Cells"

Article Title: IFN-? Upregulates Survivin and Ifi202 Expression to Induce Survival and Proliferation of Tumor-Specific T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014076

Ifi202 and survivin expression is directly regulated by the IFN-γ signaling pathway. A . IFN-γ induces ifi202 and survivin expression in T cells. T cell line 5KC was stained for IFN-γR protein level (top panel), and treated with IFN-γ for the time indicated and analyzed for STAT1 activation (middle panel) and induction of ifi202 and survivin (bottom panel). B . IFN-γ-activated STAT1 (pSTAT1) binds to the survivin promoter region. Top left panel: survivin promoter structure. Number above the bar indicates nucleotide positions relative to survivin transcription initiation site. The GAS element location and the ChIP PCR region are indicated under the bar. Bottom left panel: ChIP analysis of pSTAT1 association with the survivin promoter. 5KC cells were treated with IFN-γ for approximately 24 h and analyzed by ChIP with pSTAT1-specific mAb and survivin promoter-specific PCR. Right panel: EMSA of pSTAT1 and the GAS element of the survivin promoter. Nuclear extracts were prepared from untreated (lanes 2 and 8) and IFN-γ-treated (lanes 3–7 and 9) 5KC cells. Nuclear extracts were incubated in the presence of isotype control IgG mAb (lane 4), anti-pSTAT1 mAb (lane 5), excess cold probe (lane 6) and mutant cold probe (lane 7). Lanes 1–7: probe of survivin promoter GAS element. Lanes 8 and 9: probe of IRF8 GAS element. C . IFN-γ-activated STAT1 binds to the ifi202 5′-regulatory region. Top panel: ifi202 regulatory region structure. Number above the bar indicates nucleotide positions relative to ifi202 transcription initiation site. The GAS elements and the ChIP PCR-amplified regions are indicated under the bar. Bottom panel: ChIP analysis of pSTAT1 association with the survivin promoter. 5KC cells were treated with IFN-γ for approximately 24 h and analyzed by ChIP with pSTAT1-specific mAb and ifi202 gene-specific PCR.
Figure Legend Snippet: Ifi202 and survivin expression is directly regulated by the IFN-γ signaling pathway. A . IFN-γ induces ifi202 and survivin expression in T cells. T cell line 5KC was stained for IFN-γR protein level (top panel), and treated with IFN-γ for the time indicated and analyzed for STAT1 activation (middle panel) and induction of ifi202 and survivin (bottom panel). B . IFN-γ-activated STAT1 (pSTAT1) binds to the survivin promoter region. Top left panel: survivin promoter structure. Number above the bar indicates nucleotide positions relative to survivin transcription initiation site. The GAS element location and the ChIP PCR region are indicated under the bar. Bottom left panel: ChIP analysis of pSTAT1 association with the survivin promoter. 5KC cells were treated with IFN-γ for approximately 24 h and analyzed by ChIP with pSTAT1-specific mAb and survivin promoter-specific PCR. Right panel: EMSA of pSTAT1 and the GAS element of the survivin promoter. Nuclear extracts were prepared from untreated (lanes 2 and 8) and IFN-γ-treated (lanes 3–7 and 9) 5KC cells. Nuclear extracts were incubated in the presence of isotype control IgG mAb (lane 4), anti-pSTAT1 mAb (lane 5), excess cold probe (lane 6) and mutant cold probe (lane 7). Lanes 1–7: probe of survivin promoter GAS element. Lanes 8 and 9: probe of IRF8 GAS element. C . IFN-γ-activated STAT1 binds to the ifi202 5′-regulatory region. Top panel: ifi202 regulatory region structure. Number above the bar indicates nucleotide positions relative to ifi202 transcription initiation site. The GAS elements and the ChIP PCR-amplified regions are indicated under the bar. Bottom panel: ChIP analysis of pSTAT1 association with the survivin promoter. 5KC cells were treated with IFN-γ for approximately 24 h and analyzed by ChIP with pSTAT1-specific mAb and ifi202 gene-specific PCR.

Techniques Used: Expressing, Staining, Activation Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Incubation, Mutagenesis, Amplification

18) Product Images from "Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth"

Article Title: Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23082071

Downregulation of EGF-induced PD-L1 protein and inhibition of Akt phosphorylation in Lu99 cells treated with EGCG. ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of EGF (10 ng/mL). Numbers indicate average percentage compared with EGF-treated cells. * p
Figure Legend Snippet: Downregulation of EGF-induced PD-L1 protein and inhibition of Akt phosphorylation in Lu99 cells treated with EGCG. ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of EGF (10 ng/mL). Numbers indicate average percentage compared with EGF-treated cells. * p

Techniques Used: Inhibition, Expressing

Downregulation of IFN-γ–induced PD-L1 protein and inhibition of STAT1- and Akt-phosphorylation in A549 cells treated with (−)-epigallocatechin gallate (EGCG). ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of IFN-γ (10 ng/mL). Numbers indicate average percentage compared with IFN-γ–treated cells. * p
Figure Legend Snippet: Downregulation of IFN-γ–induced PD-L1 protein and inhibition of STAT1- and Akt-phosphorylation in A549 cells treated with (−)-epigallocatechin gallate (EGCG). ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of IFN-γ (10 ng/mL). Numbers indicate average percentage compared with IFN-γ–treated cells. * p

Techniques Used: Inhibition, Expressing

19) Product Images from "The antiviral cytokine interferon‐gamma restricts neural stem/progenitor cell proliferation through activation of STAT1 and modulation of retinoblastoma protein phosphorylation"

Article Title: The antiviral cytokine interferon‐gamma restricts neural stem/progenitor cell proliferation through activation of STAT1 and modulation of retinoblastoma protein phosphorylation

Journal: Journal of Neuroscience Research

doi: 10.1002/jnr.23987

STAT1 is required for IFNγ‐mediated inhibition of neurosphere growth. STAT1‐KO NSPCs were treated with IFNγ (1–1000 U/ml) or with heat‐inactivated IFNγ (ΔH IFNγ; 1000 U/ml) as a negative control for DIV 3 and 5 post‐IFNγ treatment A: Representative images of neurospheres imaged 5 days post‐IFNγ treatment at 2x magnification for different concentrations of IFNγ (Scale bar = 250 μm). B: Quantitation of neurosphere diameter at 3 and 5 days post‐IFNγ treatment. The longest diameter of each neurosphere was measured using Image J software. Data was collected from neurospheres in five fields/condition from three biological replicates and graphed as a percentage of untreated controls. Statistical analysis was applied by one‐way ANOVA (***p
Figure Legend Snippet: STAT1 is required for IFNγ‐mediated inhibition of neurosphere growth. STAT1‐KO NSPCs were treated with IFNγ (1–1000 U/ml) or with heat‐inactivated IFNγ (ΔH IFNγ; 1000 U/ml) as a negative control for DIV 3 and 5 post‐IFNγ treatment A: Representative images of neurospheres imaged 5 days post‐IFNγ treatment at 2x magnification for different concentrations of IFNγ (Scale bar = 250 μm). B: Quantitation of neurosphere diameter at 3 and 5 days post‐IFNγ treatment. The longest diameter of each neurosphere was measured using Image J software. Data was collected from neurospheres in five fields/condition from three biological replicates and graphed as a percentage of untreated controls. Statistical analysis was applied by one‐way ANOVA (***p

Techniques Used: Inhibition, Negative Control, Quantitation Assay, Software

IFNγ‐mediated regulation of cell cycle progression is STAT1‐dependent in NSPCs. A: STAT1‐KO NSPCs were treated with IFNγ (1, 100, 1000 U/ml) for 72h and labeled with BrdU and 7‐AAD. The BrdU and 7‐AAD intensities per cell were assayed by flow cytometry. Cell populations were gated in different phases of cell cycle (S = synthesis phase, G1 = gap phase 1, M = mitosis phase, G2 = gap phase 2). Representative plots for untreated and IFNγ‐treated NSPCs (1000 U/ml) are shown. B: Quantitation of NSPCs in each cell cycle phase. The average percentage of cells in each cell cycle phase was plotted with SEM (n = 3). Statistical analysis was applied using one‐way ANOVA (**p
Figure Legend Snippet: IFNγ‐mediated regulation of cell cycle progression is STAT1‐dependent in NSPCs. A: STAT1‐KO NSPCs were treated with IFNγ (1, 100, 1000 U/ml) for 72h and labeled with BrdU and 7‐AAD. The BrdU and 7‐AAD intensities per cell were assayed by flow cytometry. Cell populations were gated in different phases of cell cycle (S = synthesis phase, G1 = gap phase 1, M = mitosis phase, G2 = gap phase 2). Representative plots for untreated and IFNγ‐treated NSPCs (1000 U/ml) are shown. B: Quantitation of NSPCs in each cell cycle phase. The average percentage of cells in each cell cycle phase was plotted with SEM (n = 3). Statistical analysis was applied using one‐way ANOVA (**p

Techniques Used: Labeling, Flow Cytometry, Cytometry, Quantitation Assay

NSPCs activate STAT1 and STAT3 upon IFNγ stimulation. IFNγ‐treated NSPCs (100U/ml) were collected on DIV 2, 3, and 5, and lysed for western blot analysis. A: Representative blots are shown for phosphorylated STAT1 (Y701 and S727), total STAT1, phosphorylated STAT3 (Y705), and total STAT3. GAPDH is shown as the loading control. B: The fluorescence signal for each protein was quantified and normalized to GAPDH. C: For the phosphorylated STAT1 and STAT3 bands, the fluorescence signals were also normalized to the levels of the total STAT1 and STAT3, respectively. The average of 3–5 biological replicates is plotted with SEM. Statistical analysis was applied using repeated measures one‐way ANOVA with Bonferroni multiple comparisons post‐hoc analysis (****p
Figure Legend Snippet: NSPCs activate STAT1 and STAT3 upon IFNγ stimulation. IFNγ‐treated NSPCs (100U/ml) were collected on DIV 2, 3, and 5, and lysed for western blot analysis. A: Representative blots are shown for phosphorylated STAT1 (Y701 and S727), total STAT1, phosphorylated STAT3 (Y705), and total STAT3. GAPDH is shown as the loading control. B: The fluorescence signal for each protein was quantified and normalized to GAPDH. C: For the phosphorylated STAT1 and STAT3 bands, the fluorescence signals were also normalized to the levels of the total STAT1 and STAT3, respectively. The average of 3–5 biological replicates is plotted with SEM. Statistical analysis was applied using repeated measures one‐way ANOVA with Bonferroni multiple comparisons post‐hoc analysis (****p

Techniques Used: Western Blot, Fluorescence

IFNγ activates STAT3, but does not inhibit cyclin E expression or pRb phosphorylation at S795, in the absence of STAT1. STAT1‐KO NSPCs were treated with IFNγ (100U/ml) and collected on DIV 2, 3, and 5 for western blot analysis. Representative blots for phosphorylated and total STAT3 A: cyclins D1, D2, D3, E and cdk2, B: and for total pRb and associated pRb phosphorylation sites, and C: are shown. Signal intensity for each band was normalized to GAPDH as a loading control. Quantitation of signal intensity is shown as the average from 3 independent biological replicates with SEM. Statistical analysis was applied using repeated measures one‐way ANOVA with Bonferroni multiple comparisons post‐hoc analysis (*p
Figure Legend Snippet: IFNγ activates STAT3, but does not inhibit cyclin E expression or pRb phosphorylation at S795, in the absence of STAT1. STAT1‐KO NSPCs were treated with IFNγ (100U/ml) and collected on DIV 2, 3, and 5 for western blot analysis. Representative blots for phosphorylated and total STAT3 A: cyclins D1, D2, D3, E and cdk2, B: and for total pRb and associated pRb phosphorylation sites, and C: are shown. Signal intensity for each band was normalized to GAPDH as a loading control. Quantitation of signal intensity is shown as the average from 3 independent biological replicates with SEM. Statistical analysis was applied using repeated measures one‐way ANOVA with Bonferroni multiple comparisons post‐hoc analysis (*p

Techniques Used: Expressing, Western Blot, Quantitation Assay

20) Product Images from "Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease"

Article Title: Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0020131

Normal Activation of STAT1 Mutants in Stable Transfectants (A) Western blot of total protein extracts (100 μg) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, with antibodies specific for phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA or IFNG. (B) Immunofluorescence staining, with a STAT1-specific antibody, of STAT1-deficient U3C fibrosarcoma cell clones, stably cotransfected with a zeocin-resistance vector and a vector containing a WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. For (A) and (B), one experiment representative of three independent experiments is shown.
Figure Legend Snippet: Normal Activation of STAT1 Mutants in Stable Transfectants (A) Western blot of total protein extracts (100 μg) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, with antibodies specific for phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA or IFNG. (B) Immunofluorescence staining, with a STAT1-specific antibody, of STAT1-deficient U3C fibrosarcoma cell clones, stably cotransfected with a zeocin-resistance vector and a vector containing a WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. For (A) and (B), one experiment representative of three independent experiments is shown.

Techniques Used: Activation Assay, Western Blot, Clone Assay, Stable Transfection, Plasmid Preparation, Immunofluorescence, Staining

Novel STAT1 Mutations in Two Kindreds (A) STAT1 genotype and clinical phenotype of three kindreds. In kindred A, members I.1 and II.1 had tuberculosis, and III.2 and IV.1 (P1) had severe BCG disease. In kindred B, members I.1 and III. 2 (P2) were infected with M. tuberculosis and M. avium, respectively. Kindred C has been described elsewhere; II.2 (P3) developed disseminated BCG disease. Individuals with clinical disease caused by weakly virulent (BCG or M. avium ) and more virulent (M. tuberculosis) mycobacteria are indicated in black and gray, respectively, and healthy individuals are shown in white. The index cases are indicated with an arrow. Genetically affected individuals (heterozygous for any of the three STAT1 mutations) with no clinical phenotype at the time of this study are indicated by a vertical line. Known STAT1 genotypes (WT, E320Q, Q463H, L706S) are indicated under each individual, with a question mark indicating unknown genotype. (B) The human STAT1 coding region is shown, with its known pathogenic mutations. The coiled-coil domain (CC), DNA-binding domain (DNA-B), linker domain (L), SH2 domain (SH2), tail segment domain (TS), and trans -activator domain (TA) are indicated, together with their amino-acid boundaries. Tyrosine 701 (Y) is also indicated. Mutations in red are recessive mutations associated with complete STAT1 deficiency (due to a lack of STAT1 production), impaired IFNG-induced GAF activation and IFNA-induced ISGF3 activation, and a syndrome of predisposition to mycobacterial and severe viral disease in homozygous individuals. Mutations in green are associated, in heterozygous individuals, with partial STAT1 deficiency (normal STAT1 expression), impaired IFNG-induced GAS-binding activity but normal IFNA-induced ISRE-binding activity, and MSMD (predisposition to mycobacterial but not viral disease). Mutations reported for the first time in this study are indicated in italics.
Figure Legend Snippet: Novel STAT1 Mutations in Two Kindreds (A) STAT1 genotype and clinical phenotype of three kindreds. In kindred A, members I.1 and II.1 had tuberculosis, and III.2 and IV.1 (P1) had severe BCG disease. In kindred B, members I.1 and III. 2 (P2) were infected with M. tuberculosis and M. avium, respectively. Kindred C has been described elsewhere; II.2 (P3) developed disseminated BCG disease. Individuals with clinical disease caused by weakly virulent (BCG or M. avium ) and more virulent (M. tuberculosis) mycobacteria are indicated in black and gray, respectively, and healthy individuals are shown in white. The index cases are indicated with an arrow. Genetically affected individuals (heterozygous for any of the three STAT1 mutations) with no clinical phenotype at the time of this study are indicated by a vertical line. Known STAT1 genotypes (WT, E320Q, Q463H, L706S) are indicated under each individual, with a question mark indicating unknown genotype. (B) The human STAT1 coding region is shown, with its known pathogenic mutations. The coiled-coil domain (CC), DNA-binding domain (DNA-B), linker domain (L), SH2 domain (SH2), tail segment domain (TS), and trans -activator domain (TA) are indicated, together with their amino-acid boundaries. Tyrosine 701 (Y) is also indicated. Mutations in red are recessive mutations associated with complete STAT1 deficiency (due to a lack of STAT1 production), impaired IFNG-induced GAF activation and IFNA-induced ISGF3 activation, and a syndrome of predisposition to mycobacterial and severe viral disease in homozygous individuals. Mutations in green are associated, in heterozygous individuals, with partial STAT1 deficiency (normal STAT1 expression), impaired IFNG-induced GAS-binding activity but normal IFNA-induced ISRE-binding activity, and MSMD (predisposition to mycobacterial but not viral disease). Mutations reported for the first time in this study are indicated in italics.

Techniques Used: Infection, Binding Assay, Activation Assay, Expressing, Activity Assay

Normal Activation but Impaired DNA-Binding Activity of STAT1 in Heterozygous Cells from Patients (A) Western blot of total protein extracts (100 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P4) homozygous for the 1758_1759delAG mutation, probed with specific antibodies against phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. EBV-transformed B cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (B) Immunofluorescence staining with a STAT1-specific antibody of skin-derived SV40-transformed fibroblasts from a healthy control (C) and three patients under study (P1, P2, P3). Fibroblasts were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (C and E) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and the patient with complete STAT1 deficiency (P4). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 3 and 10 5 IU/ml of IFNG (C) and IFNA (E), respectively. Radiolabeled GAS (C) or ISRE (E) probes were used. (D) Quantification of four to six independent experiments by PhosphoImager SI (Molecular Dynamics, Piscataway, New Jersey, United States) using the GAS probe in response to 10 5 IU/ml of IFNG is also presented. The mean, minimum, and maximum values are expressed with respect to the positive control response (100%). For (A–C) and (E), one experiment representative of three to five independent experiments is shown.
Figure Legend Snippet: Normal Activation but Impaired DNA-Binding Activity of STAT1 in Heterozygous Cells from Patients (A) Western blot of total protein extracts (100 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P4) homozygous for the 1758_1759delAG mutation, probed with specific antibodies against phosphorylated-Tyr-701-STAT1, STAT1, and STAT3. EBV-transformed B cells were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (B) Immunofluorescence staining with a STAT1-specific antibody of skin-derived SV40-transformed fibroblasts from a healthy control (C) and three patients under study (P1, P2, P3). Fibroblasts were not stimulated (NS) or were stimulated with IFNA or IFNG (10 5 IU/ml) for 30 min. (C and E) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and the patient with complete STAT1 deficiency (P4). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 3 and 10 5 IU/ml of IFNG (C) and IFNA (E), respectively. Radiolabeled GAS (C) or ISRE (E) probes were used. (D) Quantification of four to six independent experiments by PhosphoImager SI (Molecular Dynamics, Piscataway, New Jersey, United States) using the GAS probe in response to 10 5 IU/ml of IFNG is also presented. The mean, minimum, and maximum values are expressed with respect to the positive control response (100%). For (A–C) and (E), one experiment representative of three to five independent experiments is shown.

Techniques Used: Activation Assay, Binding Assay, Activity Assay, Western Blot, Transformation Assay, Derivative Assay, Mutagenesis, Immunofluorescence, Staining, Positive Control

Pathways of IFNG-Induced GAF-Mediated Immunity and IFNA-Induced ISGF3-Mediated Immunity Patients homozygous for null STAT1 mutations [ 36 , 37 ] suffer from both mycobacterial and viral diseases. Patients heterozygous for STAT1 mutations L706S, Q463H, and E320Q suffer from mycobacterial but not viral diseases ([ 17 ] and this report).
Figure Legend Snippet: Pathways of IFNG-Induced GAF-Mediated Immunity and IFNA-Induced ISGF3-Mediated Immunity Patients homozygous for null STAT1 mutations [ 36 , 37 ] suffer from both mycobacterial and viral diseases. Patients heterozygous for STAT1 mutations L706S, Q463H, and E320Q suffer from mycobacterial but not viral diseases ([ 17 ] and this report).

Techniques Used:

Impaired DNA-Binding Activity of STAT1 Mutants in Stable Transfectants (A–D) EMSA of nuclear extracts (5 μg [A and B]; 30 μg [C and D]) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with the indicated doses of IFNG (A and B) or IFNA (C and D). We used the radiolabeled GAS probe FCGR1 (A and B) or an ISRE probe (C and D). For (A–D), one experiment representative of three to five independent experiments is shown. (E) Quantification of three independent experiments by PhosphoImager SI (Molecular Dynamics), using the ISRE probe, in response to 10 4 and 10 5 IU/ml IFNA is also presented. The mean, minimum, and maximum values are expressed with respect to the WT stable transfectant clone response (100%).
Figure Legend Snippet: Impaired DNA-Binding Activity of STAT1 Mutants in Stable Transfectants (A–D) EMSA of nuclear extracts (5 μg [A and B]; 30 μg [C and D]) from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with the indicated doses of IFNG (A and B) or IFNA (C and D). We used the radiolabeled GAS probe FCGR1 (A and B) or an ISRE probe (C and D). For (A–D), one experiment representative of three to five independent experiments is shown. (E) Quantification of three independent experiments by PhosphoImager SI (Molecular Dynamics), using the ISRE probe, in response to 10 4 and 10 5 IU/ml IFNA is also presented. The mean, minimum, and maximum values are expressed with respect to the WT stable transfectant clone response (100%).

Techniques Used: Binding Assay, Activity Assay, Clone Assay, Stable Transfection, Plasmid Preparation, Transfection

Molecular Representation of STAT1 Mutants (A) Ribbon representation of the WT STAT1 homodimer complexed with DNA. Secondary structure elements representing the β strands are shown in cyan, and the helices are shown in blue-magenta. Atoms of residues in mutated positions L706, E320, and Q463 (indicated by arrows) are shown in space-filling models. Atoms are shown in red (for oxygen), blue (for nitrogen), and gray (for carbon). (B) Magnified focus on the region containing the three mutated residues.
Figure Legend Snippet: Molecular Representation of STAT1 Mutants (A) Ribbon representation of the WT STAT1 homodimer complexed with DNA. Secondary structure elements representing the β strands are shown in cyan, and the helices are shown in blue-magenta. Atoms of residues in mutated positions L706, E320, and Q463 (indicated by arrows) are shown in space-filling models. Atoms are shown in red (for oxygen), blue (for nitrogen), and gray (for carbon). (B) Magnified focus on the region containing the three mutated residues.

Techniques Used:

Impact of STAT1 Mutations on Transcription (A) Levels of mRNA corresponding to STAT1, IFNG- and/or IFNA-inducible genes (IRF1 and ISG15) and GADP in EBV-transformed B cells from a control (C), the three affected individuals (P1, P2, and P3) and a STAT1-deficient individual (P4), or in the 2C4 parental fibrosarcoma cell line, STAT1-deficient U3C cell line, and U3C cells stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, either not stimulated (NS), or stimulated for 2 h with 10 3 of IFNG or 10 4 of IFNA for EBV-transformed B cells and 10 5 IU/ml of IFNG or IFNA for fibroblasts, as detected by Northern blotting. (B) Relative real-time PCR of IRF1, ISG15, and MX1, and cDNAs from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P5) homozygous for the 1928insA STAT1 mutation or from parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele stimulated or not stimulated with 10 5 IU/ml of IFNG or IFNA for 1 h and 2 h for EBV-transformed B cells and fibroblasts, respectively, for IRF1, and for 6 h for both cellular types for ISG15 and MX1 . Means values of duplicates of one experiment are shown with their respective standard variations.
Figure Legend Snippet: Impact of STAT1 Mutations on Transcription (A) Levels of mRNA corresponding to STAT1, IFNG- and/or IFNA-inducible genes (IRF1 and ISG15) and GADP in EBV-transformed B cells from a control (C), the three affected individuals (P1, P2, and P3) and a STAT1-deficient individual (P4), or in the 2C4 parental fibrosarcoma cell line, STAT1-deficient U3C cell line, and U3C cells stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 allele, either not stimulated (NS), or stimulated for 2 h with 10 3 of IFNG or 10 4 of IFNA for EBV-transformed B cells and 10 5 IU/ml of IFNG or IFNA for fibroblasts, as detected by Northern blotting. (B) Relative real-time PCR of IRF1, ISG15, and MX1, and cDNAs from EBV-transformed B cells derived from a healthy control (C), three patients under study (P1, P2, P3), and a patient with recessive complete STAT1 deficiency (P5) homozygous for the 1928insA STAT1 mutation or from parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele stimulated or not stimulated with 10 5 IU/ml of IFNG or IFNA for 1 h and 2 h for EBV-transformed B cells and fibroblasts, respectively, for IRF1, and for 6 h for both cellular types for ISG15 and MX1 . Means values of duplicates of one experiment are shown with their respective standard variations.

Techniques Used: Transformation Assay, Stable Transfection, Transfection, Northern Blot, Real-time Polymerase Chain Reaction, Derivative Assay, Mutagenesis, Clone Assay, Plasmid Preparation

Mechanism of Dominance of the STAT1 Alleles for GAS-Binding Activity and of Recessivity of the STAT1 Alleles for ISRE-Binding Activity (A) Western blot of total protein extracts (100 μg) from unstimulated EBV-transformed B-cell lines from a healthy control (+/+), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele), and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), using antibodies specific for STAT1 and STAT3. (B) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (+/+), P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), and P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNG. A radiolabeled GAS (FCGR1) probe was used. (C) (a) Whole-cell extracts of 10 7 STAT1-deficient U3C fibrosarcoma cell clones stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, or L706S STAT1 allele were subjected to immunoprecipitation with the following biotinylated peptides: TSFGYDKPHVLV (1), corresponding to the intracellular part of IFNGR1 around the unphosphorylated Tyr-440 residue (Y); TSFG(pTyr)DKPHVLV (2), corresponding to the intracellular part of IFNGR1 around the phosphorylated Tyr-440 residue (pTyr); and SLIG(pTyr)RPTEDSK (3), corresponding to an irrelevant peptide similar to peptide 2. (b) 20 μL of each extract was taken before immunoprecipitation, and Western blotting was performed with STAT1- and STAT3-specific antibodies. (D) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele) and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml of IFNA. A radiolabeled ISRE probe was used. (E) Immunoprecipitation with a STAT1-specific antibody, followed by Western blotting with Tyr701-phospho-STAT1–specific, Tyr690-phospho-STAT2–specific, STAT1-specific, and STAT2-specific antibodies, of total protein extracts (1 mg) from a parental fibrosarcoma cell line (2C4), a STAT2-deficient U6A fibrosarcoma cell line, and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA. For (B–E), one experiment representative of two independent experiments is shown.
Figure Legend Snippet: Mechanism of Dominance of the STAT1 Alleles for GAS-Binding Activity and of Recessivity of the STAT1 Alleles for ISRE-Binding Activity (A) Western blot of total protein extracts (100 μg) from unstimulated EBV-transformed B-cell lines from a healthy control (+/+), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele), and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), using antibodies specific for STAT1 and STAT3. (B) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (+/+), P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele), and P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNG. A radiolabeled GAS (FCGR1) probe was used. (C) (a) Whole-cell extracts of 10 7 STAT1-deficient U3C fibrosarcoma cell clones stably cotransfected with a zeocin-resistance vector and a vector containing a mock (pmock), WT, or L706S STAT1 allele were subjected to immunoprecipitation with the following biotinylated peptides: TSFGYDKPHVLV (1), corresponding to the intracellular part of IFNGR1 around the unphosphorylated Tyr-440 residue (Y); TSFG(pTyr)DKPHVLV (2), corresponding to the intracellular part of IFNGR1 around the phosphorylated Tyr-440 residue (pTyr); and SLIG(pTyr)RPTEDSK (3), corresponding to an irrelevant peptide similar to peptide 2. (b) 20 μL of each extract was taken before immunoprecipitation, and Western blotting was performed with STAT1- and STAT3-specific antibodies. (D) EMSA of nuclear extracts (5 μg) from EBV-transformed B cells derived from a healthy control (C), P4′s father (+/−) (heterozygous for the loss-of-expression, loss-of-function STAT1 1758_1759delAG allele) and P5 (−/−) (homozygous for the loss-of-expression, loss-of-function STAT1 1928insA allele). EBV-transformed B cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml of IFNA. A radiolabeled ISRE probe was used. (E) Immunoprecipitation with a STAT1-specific antibody, followed by Western blotting with Tyr701-phospho-STAT1–specific, Tyr690-phospho-STAT2–specific, STAT1-specific, and STAT2-specific antibodies, of total protein extracts (1 mg) from a parental fibrosarcoma cell line (2C4), a STAT2-deficient U6A fibrosarcoma cell line, and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H, or L706S STAT1 allele. The cells were not stimulated (NS) or were stimulated for 30 min with 10 5 IU/ml IFNA. For (B–E), one experiment representative of two independent experiments is shown.

Techniques Used: Binding Assay, Activity Assay, Western Blot, Transformation Assay, Expressing, Derivative Assay, Clone Assay, Stable Transfection, Plasmid Preparation, Immunoprecipitation

Impact of Mutant STAT1 Alleles on IFNG- and IFNA-Mediated Immunity (A) Cytokine production in the supernatant of whole blood from healthy controls (C) and patients (P1′s mother, P2, and P3) and their respective “travel” control, not stimulated (NS) or stimulated for 72 h with live BCG alone or BCG plus IL12 or IFNG. The levels of IFNG and IL12 in the supernatant were determined by enzyme-linked immunosorbent assay. One experiment representative of two independent experiments is shown. (B) Skin-derived SV40-transformed fibroblasts from a healthy control (C), the three patients under study (P1, P2, P3), a parental fibrosarcoma cell line (2C4), STAT1-deficient U3C fibrosarcoma cell line (U3C), and U3C clones stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 alleles, were infected with HSV-1 or VSV, with or without priorstimulation with IFNA (10 5 IU/ml) for 24 h. Viral titers were determined after 48 h of infection. Five independent experiments are shown for the patient's cells and three independent experiments are shown for sarcoma fibroblasts. Each assay is symbolized by a different character.
Figure Legend Snippet: Impact of Mutant STAT1 Alleles on IFNG- and IFNA-Mediated Immunity (A) Cytokine production in the supernatant of whole blood from healthy controls (C) and patients (P1′s mother, P2, and P3) and their respective “travel” control, not stimulated (NS) or stimulated for 72 h with live BCG alone or BCG plus IL12 or IFNG. The levels of IFNG and IL12 in the supernatant were determined by enzyme-linked immunosorbent assay. One experiment representative of two independent experiments is shown. (B) Skin-derived SV40-transformed fibroblasts from a healthy control (C), the three patients under study (P1, P2, P3), a parental fibrosarcoma cell line (2C4), STAT1-deficient U3C fibrosarcoma cell line (U3C), and U3C clones stably transfected with a mock (pmock), WT, E320Q, Q463H, or L706S STAT1 alleles, were infected with HSV-1 or VSV, with or without priorstimulation with IFNA (10 5 IU/ml) for 24 h. Viral titers were determined after 48 h of infection. Five independent experiments are shown for the patient's cells and three independent experiments are shown for sarcoma fibroblasts. Each assay is symbolized by a different character.

Techniques Used: Mutagenesis, Enzyme-linked Immunosorbent Assay, Derivative Assay, Transformation Assay, Clone Assay, Stable Transfection, Transfection, Infection

21) Product Images from "Intracellular Delivery of a Cell-Penetrating SOCS1 that Targets IFN-? Signaling"

Article Title: Intracellular Delivery of a Cell-Penetrating SOCS1 that Targets IFN-? Signaling

Journal: Science signaling

doi: 10.1126/scisignal.1162191

CP-SOCS1 inhibits IFN-γ– induced phosphorylation of STAT1 in AMJ2.C8 cells and BMDMs. LPS-hyporesponsive AMJ2.C8 cells ( A and B ) and BMDMs ( C and D ) were treated with diluent or the indicated concentrations of non–CP-SOCS1 or CP-SOCS1
Figure Legend Snippet: CP-SOCS1 inhibits IFN-γ– induced phosphorylation of STAT1 in AMJ2.C8 cells and BMDMs. LPS-hyporesponsive AMJ2.C8 cells ( A and B ) and BMDMs ( C and D ) were treated with diluent or the indicated concentrations of non–CP-SOCS1 or CP-SOCS1

Techniques Used:

22) Product Images from "Interferon-?-mediated Inhibition of Serum Response Factor-dependent Smooth Muscle-specific Gene Expression *"

Article Title: Interferon-?-mediated Inhibition of Serum Response Factor-dependent Smooth Muscle-specific Gene Expression *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.164863

IFNγ-STAT1 pathway reduces SRF expression and binding to CArG boxes in smooth muscle α-actin promoter. A , stellate cells were starved (0.1% serum) for 1 day and incubated with IFNγ for 2 days. Cell lysates were subjected to immunoblotting with anti-SRF antibody. B , stellate cells were starved (0.1% serum) for 1 day, then replaced with 10% serum-containing 199OR medium with or without IFNγ for 2 days. Cell lysates were subjected to immunoblotting with anti-SRF antibody. C , following transduction with the Smpro-125 luciferase reporter construct, stellate cells were incubated in 0.1% serum-containing medium for 2 days, and then the medium was changed to 10% serum-containing medium with or without IFNγ for a further 24 h. Cell lysates were assayed for luciferase activity ( n = 3; *, p
Figure Legend Snippet: IFNγ-STAT1 pathway reduces SRF expression and binding to CArG boxes in smooth muscle α-actin promoter. A , stellate cells were starved (0.1% serum) for 1 day and incubated with IFNγ for 2 days. Cell lysates were subjected to immunoblotting with anti-SRF antibody. B , stellate cells were starved (0.1% serum) for 1 day, then replaced with 10% serum-containing 199OR medium with or without IFNγ for 2 days. Cell lysates were subjected to immunoblotting with anti-SRF antibody. C , following transduction with the Smpro-125 luciferase reporter construct, stellate cells were incubated in 0.1% serum-containing medium for 2 days, and then the medium was changed to 10% serum-containing medium with or without IFNγ for a further 24 h. Cell lysates were assayed for luciferase activity ( n = 3; *, p

Techniques Used: Expressing, Binding Assay, Incubation, Transduction, Luciferase, Construct, Activity Assay

23) Product Images from "JLK1486, a Bis 8-Hydroxyquinoline-Substituted Benzylamine, Displays Cytostatic Effects in Experimental Gliomas through MyT1 and STAT1 Activation and, to a Lesser Extent, PPAR? Activation 1JLK1486, a Bis 8-Hydroxyquinoline-Substituted Benzylamine, Displays Cytostatic Effects in Experimental Gliomas through MyT1 and STAT1 Activation and, to a Lesser Extent, PPAR? Activation 1 2"

Article Title: JLK1486, a Bis 8-Hydroxyquinoline-Substituted Benzylamine, Displays Cytostatic Effects in Experimental Gliomas through MyT1 and STAT1 Activation and, to a Lesser Extent, PPAR? Activation 1JLK1486, a Bis 8-Hydroxyquinoline-Substituted Benzylamine, Displays Cytostatic Effects in Experimental Gliomas through MyT1 and STAT1 Activation and, to a Lesser Extent, PPAR? Activation 1 2

Journal: Translational Oncology

doi:

Docking of JLK1486 and Cpd30-12 in STAT1. (A) Sequence alignment of STAT1 and STAT3 (flexible residues are highlighted in red). (B) Consensus pose for Cpd30-12 is shown; electrostatic interactions with the receptor stabilize the benzoic ring and the heterocycle.
Figure Legend Snippet: Docking of JLK1486 and Cpd30-12 in STAT1. (A) Sequence alignment of STAT1 and STAT3 (flexible residues are highlighted in red). (B) Consensus pose for Cpd30-12 is shown; electrostatic interactions with the receptor stabilize the benzoic ring and the heterocycle.

Techniques Used: Sequencing

Characterization of the effects of JLK1486 on STAT1 and p21 activation. Western blot analyses of 100 nM JLK1486-mediated effects on levels of STAT1 expression and activation in human U373 GBM cells (A) and on levels of p21 expression in human U373, T98G,
Figure Legend Snippet: Characterization of the effects of JLK1486 on STAT1 and p21 activation. Western blot analyses of 100 nM JLK1486-mediated effects on levels of STAT1 expression and activation in human U373 GBM cells (A) and on levels of p21 expression in human U373, T98G,

Techniques Used: Activation Assay, Western Blot, Expressing

IC 50 in vitro growth-inhibitory concentrations of JLK1486, troglitazone and rosiglitazone in nine cancer cell lines. (A) Chemical structures of JLK1486 and Cpd30-12, a STAT1 agonist, and of troglitazone and rosiglitazone, two PPARγ agonists. (B)
Figure Legend Snippet: IC 50 in vitro growth-inhibitory concentrations of JLK1486, troglitazone and rosiglitazone in nine cancer cell lines. (A) Chemical structures of JLK1486 and Cpd30-12, a STAT1 agonist, and of troglitazone and rosiglitazone, two PPARγ agonists. (B)

Techniques Used: In Vitro

24) Product Images from "Loss of adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22"

Article Title: Loss of adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3000540

HDAC inhibition partially rescues STAT3-mediated gene transcription in Apc Min/Min organoids. (A) RT-qPCR analysis of WT and Apc Min/Min organoids treated with IL-22 (10 ng/ml) or hyper IL-6 (50 μM) for 3 hours. Data show mRNA expression of STAT3 target genes, Reg3g or Socs3 , relative to Tbp . (B) RT-qPCR analysis for WT and Apc Min/Min organoids treated with IL-22 (10 ng/ml) or IFNα (1,000 U/ml) for 3 hours. Data show the expression of STAT1 target genes, Adar and Usp18 relative to Tbp . At least 3 independent experiments were performed. * p
Figure Legend Snippet: HDAC inhibition partially rescues STAT3-mediated gene transcription in Apc Min/Min organoids. (A) RT-qPCR analysis of WT and Apc Min/Min organoids treated with IL-22 (10 ng/ml) or hyper IL-6 (50 μM) for 3 hours. Data show mRNA expression of STAT3 target genes, Reg3g or Socs3 , relative to Tbp . (B) RT-qPCR analysis for WT and Apc Min/Min organoids treated with IL-22 (10 ng/ml) or IFNα (1,000 U/ml) for 3 hours. Data show the expression of STAT1 target genes, Adar and Usp18 relative to Tbp . At least 3 independent experiments were performed. * p

Techniques Used: Inhibition, Quantitative RT-PCR, Expressing

25) Product Images from "IP3 RECEPTOR-MEDIATED Ca++-RELEASE IN NAIVE CD4 T CELLS DICTATES THEIR CYTOKINE PROGRAM 1"

Article Title: IP3 RECEPTOR-MEDIATED Ca++-RELEASE IN NAIVE CD4 T CELLS DICTATES THEIR CYTOKINE PROGRAM 1

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

doi:

Regulation of IP 3 Rs expression during the activation of CD4 and CD8 T cells (A) Freshly isolated CD4 T cells (day 0) or activated with plate bound anti-CD3 and soluble anti- CD28 mAbs for 1 or 2 days were analyzed for expression of IP 3 R1, IP 3 R2 and IP 3 R3 by Western blot analysis. STAT1 protein levels were analyzed as a loading control. Results are representative of at least three independent experiments. (B) RT-PCR was performed for IP 3 Rs in CD4 T cells that were activated as in (A). β-actin was used as house keeping gene. (C) Relative mRNA levels of IP 3 Rs in freshly isolated or CD4 T cells that were activated with anti-CD3 and anti-CD28 mAbs for 0, 6, 12, 24 and 48h were examined by real-time RT-PCR. (D) IP 3 Rs expression in unstimulated (day 0) naïve CD4 T cells, naïve cells activated with anti- CD3/anti-CD28 mAbs (day 2) and unstimulated memory CD4 T cells were examined by Western blot analysis. Densitometric analysis was performed and relative values for IP 3 R1, IP 3 R2, IP 3 R3 and STAT1 are shown (lower panel). (E) IP 3 R3 expression in unstimulated or activated memory CD4 T cells was examined by Western blot analysis. (F) IP 3 Rs expression was examined by Western blot analysis in CD4 T cells from AND TCR transgenic mice upon antigen stimulation. (G) Unstimulated or activated CD8 T cells were analyzed for expression of IP 3 Rs by RT-PCR analysis.
Figure Legend Snippet: Regulation of IP 3 Rs expression during the activation of CD4 and CD8 T cells (A) Freshly isolated CD4 T cells (day 0) or activated with plate bound anti-CD3 and soluble anti- CD28 mAbs for 1 or 2 days were analyzed for expression of IP 3 R1, IP 3 R2 and IP 3 R3 by Western blot analysis. STAT1 protein levels were analyzed as a loading control. Results are representative of at least three independent experiments. (B) RT-PCR was performed for IP 3 Rs in CD4 T cells that were activated as in (A). β-actin was used as house keeping gene. (C) Relative mRNA levels of IP 3 Rs in freshly isolated or CD4 T cells that were activated with anti-CD3 and anti-CD28 mAbs for 0, 6, 12, 24 and 48h were examined by real-time RT-PCR. (D) IP 3 Rs expression in unstimulated (day 0) naïve CD4 T cells, naïve cells activated with anti- CD3/anti-CD28 mAbs (day 2) and unstimulated memory CD4 T cells were examined by Western blot analysis. Densitometric analysis was performed and relative values for IP 3 R1, IP 3 R2, IP 3 R3 and STAT1 are shown (lower panel). (E) IP 3 R3 expression in unstimulated or activated memory CD4 T cells was examined by Western blot analysis. (F) IP 3 Rs expression was examined by Western blot analysis in CD4 T cells from AND TCR transgenic mice upon antigen stimulation. (G) Unstimulated or activated CD8 T cells were analyzed for expression of IP 3 Rs by RT-PCR analysis.

Techniques Used: Expressing, Activation Assay, Isolation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Transgenic Assay, Mouse Assay

26) Product Images from "Adipose Tissue-Derived Stem Cells Secrete CXCL5 Cytokine with Neurotrophic Effects on Cavernous Nerve Regeneration"

Article Title: Adipose Tissue-Derived Stem Cells Secrete CXCL5 Cytokine with Neurotrophic Effects on Cavernous Nerve Regeneration

Journal: The journal of sexual medicine

doi: 10.1111/j.1743-6109.2010.02128.x

CXCL5 activates JAK/STAT in Schwann cells. (A-C) Neuroblastoma cell lines BE(2)C (A) and SH-SY5Y (B) and Schwann cell line RT4-D6P2T (C) were treated with CXCL5 at 0, 5, and 50 ng/ml for 45 min and then analyzed by western blot for the expression of STAT1, phosphorylated STAT1 (pSTAT1), STAT3, and phosphorylated STAT3 (pSTAT3). β-actin served as control. (D) The ratio (in percentile) of pSTAT1 versus STAT1 expression for each treatment was determined by dividing the densitometric values of these two protein bands obtained from the western blots. Each bar represents the average of 3 independent experiments. (E) Same as in (D) except that the proteins are pSTAT3 versus STAT3.
Figure Legend Snippet: CXCL5 activates JAK/STAT in Schwann cells. (A-C) Neuroblastoma cell lines BE(2)C (A) and SH-SY5Y (B) and Schwann cell line RT4-D6P2T (C) were treated with CXCL5 at 0, 5, and 50 ng/ml for 45 min and then analyzed by western blot for the expression of STAT1, phosphorylated STAT1 (pSTAT1), STAT3, and phosphorylated STAT3 (pSTAT3). β-actin served as control. (D) The ratio (in percentile) of pSTAT1 versus STAT1 expression for each treatment was determined by dividing the densitometric values of these two protein bands obtained from the western blots. Each bar represents the average of 3 independent experiments. (E) Same as in (D) except that the proteins are pSTAT3 versus STAT3.

Techniques Used: Western Blot, Expressing

CXCL5 activates JAK/STAT time-dependently and with specificity. (A) Schwann cell line RT4-D6P2T was treated with CXCL5 at 50 ng/ml for the indicate time and then analyzed by western blot for the expression of STAT1, phosphorylated STAT1 (pSTAT1), STAT3, and phosphorylated STAT3 (pSTAT3). β-actin served as control. (B) Same as in (A) except that AG490 was added to a final concentration of 100 nM prior to the addition of CXCL5. (C) The ratio (in percentile) of pSTAT1 versus STAT1 expression for each treatment was determined by dividing the densitometric values of these two protein bands obtained from the western blots. Each bar represents the average of 3 independent experiments. (E) Same as in (C) except that the proteins are pSTAT3 versus STAT3.
Figure Legend Snippet: CXCL5 activates JAK/STAT time-dependently and with specificity. (A) Schwann cell line RT4-D6P2T was treated with CXCL5 at 50 ng/ml for the indicate time and then analyzed by western blot for the expression of STAT1, phosphorylated STAT1 (pSTAT1), STAT3, and phosphorylated STAT3 (pSTAT3). β-actin served as control. (B) Same as in (A) except that AG490 was added to a final concentration of 100 nM prior to the addition of CXCL5. (C) The ratio (in percentile) of pSTAT1 versus STAT1 expression for each treatment was determined by dividing the densitometric values of these two protein bands obtained from the western blots. Each bar represents the average of 3 independent experiments. (E) Same as in (C) except that the proteins are pSTAT3 versus STAT3.

Techniques Used: Western Blot, Expressing, Concentration Assay

27) Product Images from "Differential Responses of Human Dendritic Cells to Live or Inactivated Staphylococcus aureus: Impact on Cytokine Production and T Helper Expansion"

Article Title: Differential Responses of Human Dendritic Cells to Live or Inactivated Staphylococcus aureus: Impact on Cytokine Production and T Helper Expansion

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02622

Expression of phosphorylated STAT1/2 and IRF7 in DC stimulated with live and UVI-USA300. DC were left untreated (Ctrl) or stimulated with live and UVI-USA300 for 5 and 24 h. Cell lysates were analyzed by Western blot to detect the expression and the phosphorylation of the indicated proteins. β-actin levels were analyzed as control for protein loading.
Figure Legend Snippet: Expression of phosphorylated STAT1/2 and IRF7 in DC stimulated with live and UVI-USA300. DC were left untreated (Ctrl) or stimulated with live and UVI-USA300 for 5 and 24 h. Cell lysates were analyzed by Western blot to detect the expression and the phosphorylation of the indicated proteins. β-actin levels were analyzed as control for protein loading.

Techniques Used: Expressing, Western Blot

28) Product Images from "Brain-derived neurotrophic factor promotes nerve regeneration by activating the JAK/STAT pathway in Schwann cells"

Article Title: Brain-derived neurotrophic factor promotes nerve regeneration by activating the JAK/STAT pathway in Schwann cells

Journal: Translational Andrology and Urology

doi: 10.21037/tau.2016.02.03

Time response of JAK/STAT pathway activation in RT4-D6P2T after BDNF administration. BDNF (100 pM) was administered to treat rat Schwann cells RT4-D6P2T at 0 min, 10 min, 30 min, 60 min, 120 min and 24 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 30 min (lane 3) (*P
Figure Legend Snippet: Time response of JAK/STAT pathway activation in RT4-D6P2T after BDNF administration. BDNF (100 pM) was administered to treat rat Schwann cells RT4-D6P2T at 0 min, 10 min, 30 min, 60 min, 120 min and 24 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 30 min (lane 3) (*P

Techniques Used: Activation Assay

BDNF activates JAK/STAT directly. The HSC and RT4-D6P2T cells were validated by immunofluorescence staining. (A) The cellular markers of Schwann cells, S100 and p75, were expressed in cells in the cytoplasm represented as green (FTIC) and nucleus as blue (DAPI) (×100); (B,C) different doses of BDNF were applied to treat BE( 2 )-C, SH-SY5Y and RT4-D6P2T (0, 1, 10, 100 pM) for 30 minutes. The phosphorylation of STAT3/STAT1 was extensively enhanced by BDNF at a dose of (100 pM) in RT4-D6P2T cells (*P
Figure Legend Snippet: BDNF activates JAK/STAT directly. The HSC and RT4-D6P2T cells were validated by immunofluorescence staining. (A) The cellular markers of Schwann cells, S100 and p75, were expressed in cells in the cytoplasm represented as green (FTIC) and nucleus as blue (DAPI) (×100); (B,C) different doses of BDNF were applied to treat BE( 2 )-C, SH-SY5Y and RT4-D6P2T (0, 1, 10, 100 pM) for 30 minutes. The phosphorylation of STAT3/STAT1 was extensively enhanced by BDNF at a dose of (100 pM) in RT4-D6P2T cells (*P

Techniques Used: Immunofluorescence, Staining

Time response of JAK/STAT pathway activation in HSCs after BDNF treatment. BDNF (100 pM) was administered to treat the human Schwann cells at 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr and 72 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 60 min (lane 4) (*P
Figure Legend Snippet: Time response of JAK/STAT pathway activation in HSCs after BDNF treatment. BDNF (100 pM) was administered to treat the human Schwann cells at 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr and 72 hr. STAT3/STAT1 were activated at 10 min after the treatment with BDNF (lane 2), and the phosphorylation level peaked at 60 min (lane 4) (*P

Techniques Used: Activation Assay

Mechanism of JAK/STAT re-activation by BDNF in HSCs. BDNF (100 pM) was administered to treat the human Schwann cell HSCs for 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr, 48 hr, and 72 hr. (A) JAK2 was activated by BDNF treatment and possessed two phosphorylation peaks, which was similar to the response of STAT3/STAT1; (B) the phosphorylation levels of JAK2, STAT1 and STAT3 were presented as a ratio of phosphorylated form to total expressed forms. P1: phosphorylation early peak. P2: phosphorylation late peak. The cell culture mediums from the above treatment were applied to assay the level of cytokines secreted from HSCs by ELISA; (C) 1 hr after the BDNF treatment, HSCs began to secrete OSM-M at levels reaching ~20±0.8 pg/mL at 24 hr ( # P > 0.05); (D) BDNF significantly increased the secretion of IL6 from HSCs after 1 hr of the treatment and reached ~360.9±74 pg/mL at 72 hr (*P
Figure Legend Snippet: Mechanism of JAK/STAT re-activation by BDNF in HSCs. BDNF (100 pM) was administered to treat the human Schwann cell HSCs for 0 min, 10 min, 30 min, 60 min, 120 min, 24 hr, 48 hr, and 72 hr. (A) JAK2 was activated by BDNF treatment and possessed two phosphorylation peaks, which was similar to the response of STAT3/STAT1; (B) the phosphorylation levels of JAK2, STAT1 and STAT3 were presented as a ratio of phosphorylated form to total expressed forms. P1: phosphorylation early peak. P2: phosphorylation late peak. The cell culture mediums from the above treatment were applied to assay the level of cytokines secreted from HSCs by ELISA; (C) 1 hr after the BDNF treatment, HSCs began to secrete OSM-M at levels reaching ~20±0.8 pg/mL at 24 hr ( # P > 0.05); (D) BDNF significantly increased the secretion of IL6 from HSCs after 1 hr of the treatment and reached ~360.9±74 pg/mL at 72 hr (*P

Techniques Used: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

29) Product Images from "Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia"

Article Title: Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067134

Increased endogenous STAT1 phosphorylation in human Evi1 overexpressed leukemic cell lines. a) Western blot analysis using anti-total-STAT1 antibody. Lane 1 from left shows total STAT1 protein expression level in Kasumi 1 cells. Lane 2 shows total STAT1 protein level in Kasumi 3 cells. Evi1 overexpressed myeloid leukemic cells demonstrate a higher baseline of STAT1 protein, consistent with our mRNA findings. b) Western blot analysis using anti-phospho-STAT1 antibody. Lane 1 from left shows endogenous phosphorylated STAT1 protein expression level in Kasumi 1 cells (human leukemic cell line without Evi1 expression. Lane 2 shows the STAT1 protein level in Kasumi 3 cells. c) Beta actin loading control.
Figure Legend Snippet: Increased endogenous STAT1 phosphorylation in human Evi1 overexpressed leukemic cell lines. a) Western blot analysis using anti-total-STAT1 antibody. Lane 1 from left shows total STAT1 protein expression level in Kasumi 1 cells. Lane 2 shows total STAT1 protein level in Kasumi 3 cells. Evi1 overexpressed myeloid leukemic cells demonstrate a higher baseline of STAT1 protein, consistent with our mRNA findings. b) Western blot analysis using anti-phospho-STAT1 antibody. Lane 1 from left shows endogenous phosphorylated STAT1 protein expression level in Kasumi 1 cells (human leukemic cell line without Evi1 expression. Lane 2 shows the STAT1 protein level in Kasumi 3 cells. c) Beta actin loading control.

Techniques Used: Western Blot, Expressing

Significant deregulation of the Jak-Stat signaling pathway. Numerous genes involved in the Jak-Stat pathway were found to be aberrantly expressed in both the EVI1 leukemic cell lines. The x-axis is the significantly downregulated gene and the y-axis denotes reads per million on RNA-Seq analysis. a ) In DA-1 Evi1 overexpressed cells, Socs1 had a 5.7-fold decrease (p = 0.001), Osm had a 13-fold decrease (p = 0.0003), Myc had a 2.5-fold decrease (p = 0.04), Il6 had a 2.8-fold decrease (p = 0.02), Csf2rb had a 3.2-fold decrease (p = 0.02). b) In NFS-60 Evi1 overexpressed cells, Stat5 had a 2.1-fold increase (p = 0.01), Stat1 had a 2.0-fold increase (p = 0.02), Socs3 had a 3.7-fold decrease (p = 0.03), Socs2 had a 3.0-fold decrease (p = 0.01), Socs1 had a 4.5-fold decrease (p = 0.02). c) In NFS-60 Evi1 overexpressed cells, Pik3c2g had a 2.8-fold increase (p = 0.04) Pim1 had 2.2-fold decrease (p = 0.01), Osm had a 1.8-fold decrease (p = 0.04), Il4r had a 2.3-fold decrease (p = 0.01), Crebbp had a 1.9-fold increase (p = 0.05), Cish had a 2.0-fold decrease (p = 0.05).
Figure Legend Snippet: Significant deregulation of the Jak-Stat signaling pathway. Numerous genes involved in the Jak-Stat pathway were found to be aberrantly expressed in both the EVI1 leukemic cell lines. The x-axis is the significantly downregulated gene and the y-axis denotes reads per million on RNA-Seq analysis. a ) In DA-1 Evi1 overexpressed cells, Socs1 had a 5.7-fold decrease (p = 0.001), Osm had a 13-fold decrease (p = 0.0003), Myc had a 2.5-fold decrease (p = 0.04), Il6 had a 2.8-fold decrease (p = 0.02), Csf2rb had a 3.2-fold decrease (p = 0.02). b) In NFS-60 Evi1 overexpressed cells, Stat5 had a 2.1-fold increase (p = 0.01), Stat1 had a 2.0-fold increase (p = 0.02), Socs3 had a 3.7-fold decrease (p = 0.03), Socs2 had a 3.0-fold decrease (p = 0.01), Socs1 had a 4.5-fold decrease (p = 0.02). c) In NFS-60 Evi1 overexpressed cells, Pik3c2g had a 2.8-fold increase (p = 0.04) Pim1 had 2.2-fold decrease (p = 0.01), Osm had a 1.8-fold decrease (p = 0.04), Il4r had a 2.3-fold decrease (p = 0.01), Crebbp had a 1.9-fold increase (p = 0.05), Cish had a 2.0-fold decrease (p = 0.05).

Techniques Used: RNA Sequencing Assay, Chromogenic In Situ Hybridization

Pathway diagram of the KEGG Jak-Stat pathway using DAVID analysis. EVI1 binds to the majority (78%) of major genes involved in the regulation of the Jak-Stat signaling pathway. These include Il10, Il10ra, Il6, Il6ra, Cbl, Jak1, Pias2, Stat1, Stat4, Stat5, Stat6, Grb, Sos2, Akt1, Akt2, Socs1,Pim1, Ccnd1, Ccnd2, Ccnd3, Myc, and Spred1 . EVI1 binds to an AGGAAG ETS-like motif which is present in the promoter region of all of these Jak-Stat pathway genes. Illustration taken from DAVID website http://david.abcc.ncifcrf.gov/ .
Figure Legend Snippet: Pathway diagram of the KEGG Jak-Stat pathway using DAVID analysis. EVI1 binds to the majority (78%) of major genes involved in the regulation of the Jak-Stat signaling pathway. These include Il10, Il10ra, Il6, Il6ra, Cbl, Jak1, Pias2, Stat1, Stat4, Stat5, Stat6, Grb, Sos2, Akt1, Akt2, Socs1,Pim1, Ccnd1, Ccnd2, Ccnd3, Myc, and Spred1 . EVI1 binds to an AGGAAG ETS-like motif which is present in the promoter region of all of these Jak-Stat pathway genes. Illustration taken from DAVID website http://david.abcc.ncifcrf.gov/ .

Techniques Used:

30) Product Images from "Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity ▿"

Article Title: Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00011-10

The Ni-CE P protein is defective for cytoplasmic localization and for its capacity to inhibit nuclear import of IFN-activated STAT1. (A) Vero cells were transfected to express the indicated GFP-tagged P protein (green) and, 18 h later, were treated with or without IFN-α for 1 h. The cells were fixed with formaldehyde and methanol and immunostained for STAT1 (red) before being analyzed by CLSM. (B, C) Images such as those shown in panel A were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 130, combined data from 3 separate assays) for STAT1 (B) or GFP-tagged P protein (C). Ni, Nishigahara.
Figure Legend Snippet: The Ni-CE P protein is defective for cytoplasmic localization and for its capacity to inhibit nuclear import of IFN-activated STAT1. (A) Vero cells were transfected to express the indicated GFP-tagged P protein (green) and, 18 h later, were treated with or without IFN-α for 1 h. The cells were fixed with formaldehyde and methanol and immunostained for STAT1 (red) before being analyzed by CLSM. (B, C) Images such as those shown in panel A were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 130, combined data from 3 separate assays) for STAT1 (B) or GFP-tagged P protein (C). Ni, Nishigahara.

Techniques Used: Transfection, Confocal Laser Scanning Microscopy, Fluorescence

The NES in RV P protein plays an important role in its IFN antagonism. (A) In order to restore the NES activity to the Ni-CE P protein [producing Ni-CE P(NES + )-GFP], Pro-to-Leu substitutions were introduced into Ni-CE P-GFP at positions 56 and 58. (B) To compare the subcellular localization of Ni-CE P(NES + )-GFP with that of Ni-CE P-GFP, SK-N-SH cells were transfected with plasmid pEGFP-N1 expressing the respective protein and images were collected 24 h posttransfection. (C) Vero cells were transfected to express Ni-CE P-GFP or Ni-CE P(NES + )-GFP (green) and treated with or without IFN-α before being fixed and immunostained for STAT1 (red) and analyzed by CLSM. (D, E) Images such as those shown in panel C were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 30) for GFP-tagged P protein (D) or STAT1 (E). (F) SK-N-SH cells were transfected with the ISRE reporter and the control plasmids, together with the pEGFP-N1 plasmid expressing Ni-GFP, Ni-CE GFP, or Ni-CE P(NES + )-GFP. At 24 h posttransfection, the cells were treated with IFN-α (2,000 U/ml) for 6 h and the cell lysates were subjected to the dual luciferase assay. GL, firefly luciferase activity; RL, Renilla luciferase activity; ns, not significant ( P ≥ 0.05).
Figure Legend Snippet: The NES in RV P protein plays an important role in its IFN antagonism. (A) In order to restore the NES activity to the Ni-CE P protein [producing Ni-CE P(NES + )-GFP], Pro-to-Leu substitutions were introduced into Ni-CE P-GFP at positions 56 and 58. (B) To compare the subcellular localization of Ni-CE P(NES + )-GFP with that of Ni-CE P-GFP, SK-N-SH cells were transfected with plasmid pEGFP-N1 expressing the respective protein and images were collected 24 h posttransfection. (C) Vero cells were transfected to express Ni-CE P-GFP or Ni-CE P(NES + )-GFP (green) and treated with or without IFN-α before being fixed and immunostained for STAT1 (red) and analyzed by CLSM. (D, E) Images such as those shown in panel C were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 30) for GFP-tagged P protein (D) or STAT1 (E). (F) SK-N-SH cells were transfected with the ISRE reporter and the control plasmids, together with the pEGFP-N1 plasmid expressing Ni-GFP, Ni-CE GFP, or Ni-CE P(NES + )-GFP. At 24 h posttransfection, the cells were treated with IFN-α (2,000 U/ml) for 6 h and the cell lysates were subjected to the dual luciferase assay. GL, firefly luciferase activity; RL, Renilla luciferase activity; ns, not significant ( P ≥ 0.05).

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Confocal Laser Scanning Microscopy, Fluorescence, Luciferase

The subcellular localization of STAT1 in RV-infected SK-N-SH cells differs between the viral strains. (A) The cells were inoculated with each strain at an MOI of 0.01 and then were treated with IFN-α (4,000 U/ml) for 30 min at 24 h p.i. The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min before being immunostained for STAT1 (green) and RV N protein (red) and analyzed by CLSM. (B) Images such as those shown in panel A were used to calculate the ratios of nuclear to cytoplasmic fluorescence (Fn/c) of STAT1, which are shown as the means ± standard errors of the means of the results from > 30 images. ns, not significant ( P ≥ 0.05); Ni, Nishigahara.
Figure Legend Snippet: The subcellular localization of STAT1 in RV-infected SK-N-SH cells differs between the viral strains. (A) The cells were inoculated with each strain at an MOI of 0.01 and then were treated with IFN-α (4,000 U/ml) for 30 min at 24 h p.i. The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min before being immunostained for STAT1 (green) and RV N protein (red) and analyzed by CLSM. (B) Images such as those shown in panel A were used to calculate the ratios of nuclear to cytoplasmic fluorescence (Fn/c) of STAT1, which are shown as the means ± standard errors of the means of the results from > 30 images. ns, not significant ( P ≥ 0.05); Ni, Nishigahara.

Techniques Used: Infection, Confocal Laser Scanning Microscopy, Fluorescence

Both the Nishigahara (Ni) and Ni-CE P proteins physically interact with STAT1. (A) Yeast cells (L40 strain) were cotransformed with plasmid pLex-CVS P, -Ni P, or -Ni-CE P and plasmid pGAD-STAT1 (+) or the empty pGAD plasmid (−). The P protein-STAT1 interaction was assessed by the appearance of blue colonies in the presence of X-Gal on a plate lacking Trp and Leu (upper panel) and by the expression of the His3 reporter gene on a plate lacking Trp, Leu, and His (lower panel). (B) SK-N-SH cells were inoculated with strain Ni-CE or CE(NiP) at an MOI of 0.1. At 18 h p.i., the cells were treated with IFN-α for 2 h before being lysed in RIPA buffer. The cell lysates were subjected to co-IP analysis with an anti-STAT1 antibody or control rabbit IgG. The precipitates and total lysate (input) were analyzed by Western blotting (WB). The asterisk represents an additional band probably resulting from binding of antibodies used for Western blotting to protein A/G. α-Tubulin, alpha-tubulin.
Figure Legend Snippet: Both the Nishigahara (Ni) and Ni-CE P proteins physically interact with STAT1. (A) Yeast cells (L40 strain) were cotransformed with plasmid pLex-CVS P, -Ni P, or -Ni-CE P and plasmid pGAD-STAT1 (+) or the empty pGAD plasmid (−). The P protein-STAT1 interaction was assessed by the appearance of blue colonies in the presence of X-Gal on a plate lacking Trp and Leu (upper panel) and by the expression of the His3 reporter gene on a plate lacking Trp, Leu, and His (lower panel). (B) SK-N-SH cells were inoculated with strain Ni-CE or CE(NiP) at an MOI of 0.1. At 18 h p.i., the cells were treated with IFN-α for 2 h before being lysed in RIPA buffer. The cell lysates were subjected to co-IP analysis with an anti-STAT1 antibody or control rabbit IgG. The precipitates and total lysate (input) were analyzed by Western blotting (WB). The asterisk represents an additional band probably resulting from binding of antibodies used for Western blotting to protein A/G. α-Tubulin, alpha-tubulin.

Techniques Used: Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay, Western Blot, Binding Assay

31) Product Images from "The T cell STAT signaling network is reprogrammed within hours of bacteremia via secondary signals"

Article Title: The T cell STAT signaling network is reprogrammed within hours of bacteremia via secondary signals

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

doi: 10.4049/jimmunol.0803666

The Stat1 response to IL-6 was reduced in peripheral blood T cells from E. coli -challenged mice
Figure Legend Snippet: The Stat1 response to IL-6 was reduced in peripheral blood T cells from E. coli -challenged mice

Techniques Used: Mouse Assay

Cytokine-induced phosphorylation of Stat1 is repressed post-bacteremia
Figure Legend Snippet: Cytokine-induced phosphorylation of Stat1 is repressed post-bacteremia

Techniques Used:

32) Product Images from "Hypoxia Modulates A431 Cellular Pathways Association to Tumor Radioresistance and Enhanced Migration Revealed by Comprehensive Proteomic and Functional Studies *"

Article Title: Hypoxia Modulates A431 Cellular Pathways Association to Tumor Radioresistance and Enhanced Migration Revealed by Comprehensive Proteomic and Functional Studies *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M112.018325

Confirmation of STAT1 pathway suppressed by hypoxia and validation of stable cell lines with STAT1α overexpression in A431; A , Change patterns of STAT1 and its downstream proteins because of hypoxia or reoxygenation; B , The down-regulation of STAT1 and pSTAT1α (S727) was confirmed by WB. C , The decrease of functional STAT1 represses the transcription of its downstream genes. D , The two positive stable cell lines with STAT1α overexpression were confirmed by WB using anti-FLAG, STAT1 and pSTAT1α (S727) antibodies. E , The activation of downstream genes of STAT1 in the stable cell lines indicated that the cell lines indeed have more active STAT1α.
Figure Legend Snippet: Confirmation of STAT1 pathway suppressed by hypoxia and validation of stable cell lines with STAT1α overexpression in A431; A , Change patterns of STAT1 and its downstream proteins because of hypoxia or reoxygenation; B , The down-regulation of STAT1 and pSTAT1α (S727) was confirmed by WB. C , The decrease of functional STAT1 represses the transcription of its downstream genes. D , The two positive stable cell lines with STAT1α overexpression were confirmed by WB using anti-FLAG, STAT1 and pSTAT1α (S727) antibodies. E , The activation of downstream genes of STAT1 in the stable cell lines indicated that the cell lines indeed have more active STAT1α.

Techniques Used: Stable Transfection, Over Expression, Western Blot, Functional Assay, Activation Assay

33) Product Images from "Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth"

Article Title: Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23082071

Downregulation of IFN-γ–induced PD-L1 protein and inhibition of STAT1- and Akt-phosphorylation in A549 cells treated with (−)-epigallocatechin gallate (EGCG). ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of IFN-γ (10 ng/mL). Numbers indicate average percentage compared with IFN-γ–treated cells. * p
Figure Legend Snippet: Downregulation of IFN-γ–induced PD-L1 protein and inhibition of STAT1- and Akt-phosphorylation in A549 cells treated with (−)-epigallocatechin gallate (EGCG). ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of IFN-γ (10 ng/mL). Numbers indicate average percentage compared with IFN-γ–treated cells. * p

Techniques Used: Inhibition, Expressing

Downregulation of EGF-induced PD-L1 protein and inhibition of Akt phosphorylation in Lu99 cells treated with EGCG. ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of EGF (10 ng/mL). Numbers indicate average percentage compared with EGF-treated cells. * p
Figure Legend Snippet: Downregulation of EGF-induced PD-L1 protein and inhibition of Akt phosphorylation in Lu99 cells treated with EGCG. ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of EGF (10 ng/mL). Numbers indicate average percentage compared with EGF-treated cells. * p

Techniques Used: Inhibition, Expressing

34) Product Images from "A biallelic mutation in IL6ST encoding the GP130 co-receptor causes immunodeficiency and craniosynostosis"

Article Title: A biallelic mutation in IL6ST encoding the GP130 co-receptor causes immunodeficiency and craniosynostosis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161810

The GP130 p.N404Y variant causes a defective acute phase response, and P1 primary fibroblasts can be rescued by lentiviral transduction of WT GP130. (A) Cellular and biochemical response to recurrent chest infections and cellulitis in patient GP130 p.N404Y. White blood cell count (WBC), neutrophils, CRP, and fibrinogen measurements are shown at different time points. Black/red lines indicate 3rd/97th percentile or lower/upper limit of the normal range, respectively. (B) CRISPR/Cas9-mediated KO of GP130 in human hepatoma Hep3B cells results in absent GP130 surface expression. (C) IL-6–mediated production of fibrinogen is GP130 dependent. Hep3B GP130-KO cells were stimulated with IL-6 for 24 h, and expression of the fibrinogen α chain ( FGA ; left) and fibrinogen β chain ( FGB ; right) was determined by quantitative PCR. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with unstimulated cells. Data represent summary results from four independent experiments (mean ± SEM). (D) Hep3B GP130-KO cells were transiently transfected with GP130 WT, GP130 p.N404Y-mutant plasmid, and FGA (left) and FGB (right) gene expression was analyzed after 24 h of IL-6 stimulation. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with the unstimulated vector control. Data represent pooled results from four independent experiments (mean ± SEM). (E) Fibroblasts of a healthy donor and P1 were stimulated with the indicated concentrations (ng/ml) of IL-6, IL-11, IL-27, OSM, and LIF. The levels of phospho-STAT3 (p-STAT3) were determined after 15-min stimulation by Phosflow. Titration curves are representative of two independent experiments. Curves are fitted by nonlinear/linear regression analysis. (F) Healthy donor and patient GP130 p.N404Y fibroblasts were treated as in E, and STAT1 phosphorylation (p-STAT1) was analyzed using flow cytometry. Titration curves are representative of two independent experiements. MFI, mean fluorescence intensity. (G) Lentiviral transduction of GP130 WT reconstitutes STAT3 phosphorylation in primary fibroblasts with p.N404Y variant. Fibroblasts were stimulated with 30 ng/ml IL-6 and 50 ng/ml IL-11 for 15 min. Quantification is based on four independent experiments. HD, healthy donor; LV, lentiviral. Differences were determined by Mann-Whitney U test. *, P
Figure Legend Snippet: The GP130 p.N404Y variant causes a defective acute phase response, and P1 primary fibroblasts can be rescued by lentiviral transduction of WT GP130. (A) Cellular and biochemical response to recurrent chest infections and cellulitis in patient GP130 p.N404Y. White blood cell count (WBC), neutrophils, CRP, and fibrinogen measurements are shown at different time points. Black/red lines indicate 3rd/97th percentile or lower/upper limit of the normal range, respectively. (B) CRISPR/Cas9-mediated KO of GP130 in human hepatoma Hep3B cells results in absent GP130 surface expression. (C) IL-6–mediated production of fibrinogen is GP130 dependent. Hep3B GP130-KO cells were stimulated with IL-6 for 24 h, and expression of the fibrinogen α chain ( FGA ; left) and fibrinogen β chain ( FGB ; right) was determined by quantitative PCR. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with unstimulated cells. Data represent summary results from four independent experiments (mean ± SEM). (D) Hep3B GP130-KO cells were transiently transfected with GP130 WT, GP130 p.N404Y-mutant plasmid, and FGA (left) and FGB (right) gene expression was analyzed after 24 h of IL-6 stimulation. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with the unstimulated vector control. Data represent pooled results from four independent experiments (mean ± SEM). (E) Fibroblasts of a healthy donor and P1 were stimulated with the indicated concentrations (ng/ml) of IL-6, IL-11, IL-27, OSM, and LIF. The levels of phospho-STAT3 (p-STAT3) were determined after 15-min stimulation by Phosflow. Titration curves are representative of two independent experiments. Curves are fitted by nonlinear/linear regression analysis. (F) Healthy donor and patient GP130 p.N404Y fibroblasts were treated as in E, and STAT1 phosphorylation (p-STAT1) was analyzed using flow cytometry. Titration curves are representative of two independent experiements. MFI, mean fluorescence intensity. (G) Lentiviral transduction of GP130 WT reconstitutes STAT3 phosphorylation in primary fibroblasts with p.N404Y variant. Fibroblasts were stimulated with 30 ng/ml IL-6 and 50 ng/ml IL-11 for 15 min. Quantification is based on four independent experiments. HD, healthy donor; LV, lentiviral. Differences were determined by Mann-Whitney U test. *, P

Techniques Used: Variant Assay, Transduction, Cell Counting, CRISPR, Expressing, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation, Titration, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

The GP130 p.N404Y substitution causes defective signaling by IL-11, IL-6, IL-27, OSM, and LIF. (A–E) HEK293 GP130-KO cells were transfected with empty vector control or plasmids encoding GP130 WT or the patient variant p.N404Y. Cells were stimulated with the indicated concentrations (ng/ml) of IL-11 (A), IL-6 (B), IL-27 (C), OSM (D), or LIF (E) for 15 min and analyzed for STAT3 phosphorylation (p-STAT3) by Phosflow. For assessment of IL-11 and IL-6 signaling, cells were cotransfected with plasmids encoding IL-11RA and IL-6RA, respectively. Co-transfection with GFP allowed gating on successfully transfected cells. Representative titration curves (on the left in each panel) are shown for each ligand and are representative of two independent experiments. Curve fitting is by nonlinear regression. Quantification (on the right in each panel) is based on four to six independent experiments per cytokine at one concentration (IL-11, 1 ng/ml; IL-6, IL-27, OSM, and LIF, all 100 ng/ml). (F) Experiments with HEK293 GP130-KO cells performed as in A–C. Cells were assayed for phospho-STAT1 (p-STAT1). Titration curves are representative of two independent experiments. MFI, mean fluorescence intensity. (G) Immunofluorescence staining of HEK293 GP130-KO cells, plated in chamber slides and transfected as in A. Cells were analyzed for STAT3 nuclear translocation using confocal microscopy. Bars, 50 µm. Images on the right are merged and magnified. Images are representative for three independent experiments. (H) HEK293 cells were cotransfected with luciferase (Luc) reporters, GP130 variants, and IL-11RA or IL-6RA expression vectors, respectively. After 24 h, cells were stimulated with 1 ng/ml IL-11 (top) or 0.5 ng/ml IL-6 (bottom) for 6 h, and induction of STAT3 reporter (relative to constitutively expressed Renilla luciferase) was determined. Results are expressed as fold-induction compared with unstimulated vector control and are pooled data from three independent experiments with three to six technical replicates each. Data represent mean with SEM. Differences were investigated by Mann-Whitney U test. **, P
Figure Legend Snippet: The GP130 p.N404Y substitution causes defective signaling by IL-11, IL-6, IL-27, OSM, and LIF. (A–E) HEK293 GP130-KO cells were transfected with empty vector control or plasmids encoding GP130 WT or the patient variant p.N404Y. Cells were stimulated with the indicated concentrations (ng/ml) of IL-11 (A), IL-6 (B), IL-27 (C), OSM (D), or LIF (E) for 15 min and analyzed for STAT3 phosphorylation (p-STAT3) by Phosflow. For assessment of IL-11 and IL-6 signaling, cells were cotransfected with plasmids encoding IL-11RA and IL-6RA, respectively. Co-transfection with GFP allowed gating on successfully transfected cells. Representative titration curves (on the left in each panel) are shown for each ligand and are representative of two independent experiments. Curve fitting is by nonlinear regression. Quantification (on the right in each panel) is based on four to six independent experiments per cytokine at one concentration (IL-11, 1 ng/ml; IL-6, IL-27, OSM, and LIF, all 100 ng/ml). (F) Experiments with HEK293 GP130-KO cells performed as in A–C. Cells were assayed for phospho-STAT1 (p-STAT1). Titration curves are representative of two independent experiments. MFI, mean fluorescence intensity. (G) Immunofluorescence staining of HEK293 GP130-KO cells, plated in chamber slides and transfected as in A. Cells were analyzed for STAT3 nuclear translocation using confocal microscopy. Bars, 50 µm. Images on the right are merged and magnified. Images are representative for three independent experiments. (H) HEK293 cells were cotransfected with luciferase (Luc) reporters, GP130 variants, and IL-11RA or IL-6RA expression vectors, respectively. After 24 h, cells were stimulated with 1 ng/ml IL-11 (top) or 0.5 ng/ml IL-6 (bottom) for 6 h, and induction of STAT3 reporter (relative to constitutively expressed Renilla luciferase) was determined. Results are expressed as fold-induction compared with unstimulated vector control and are pooled data from three independent experiments with three to six technical replicates each. Data represent mean with SEM. Differences were investigated by Mann-Whitney U test. **, P

Techniques Used: Transfection, Plasmid Preparation, Variant Assay, Cotransfection, Titration, Concentration Assay, Fluorescence, Immunofluorescence, Staining, Translocation Assay, Confocal Microscopy, Luciferase, Expressing, MANN-WHITNEY

35) Product Images from "Anti-Inflammatory Effect of Quercetagetin, an Active Component of Immature Citrus unshiu, in HaCaT Human Keratinocytes"

Article Title: Anti-Inflammatory Effect of Quercetagetin, an Active Component of Immature Citrus unshiu, in HaCaT Human Keratinocytes

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2013.001

Effect of quercetagetin on STAT1 signal related with TARC and MDC expressions in IFN-γ- and TNF-α-stimulated HaCaT human keratinocytes. (A) HaCaT cells (5.0×10 5 cells/ml) were pre-incubated for 18 hr in unsupplemented culture medium. The cells were then stimulated with IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) in the presence of Jak I (Jak1/2 inhibitor) with indicated concentrations for 24 hr. The measurements of TARC and MDC were determined from culture supernatant by the ELISA in triplicate. Error bars indicate ± S.D. * p
Figure Legend Snippet: Effect of quercetagetin on STAT1 signal related with TARC and MDC expressions in IFN-γ- and TNF-α-stimulated HaCaT human keratinocytes. (A) HaCaT cells (5.0×10 5 cells/ml) were pre-incubated for 18 hr in unsupplemented culture medium. The cells were then stimulated with IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) in the presence of Jak I (Jak1/2 inhibitor) with indicated concentrations for 24 hr. The measurements of TARC and MDC were determined from culture supernatant by the ELISA in triplicate. Error bars indicate ± S.D. * p

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

Related Articles

Staining:

Article Title: Decreased STAT5 phosphorylation and GATA-3 expression in NOX2 deficient T cells: Role in T helper development
Article Snippet: .. Phosphorylated STAT levels were determined by intracellular staining with antibodies specific for pY701-STAT1 (4a), pY705-STAT3 (4/P-STAT3), pY693-STAT4 (38/p-Stat4), pY694-STAT5 (47), and pY641-STAT6 (J71-773.58.11) (all BD Pharmingen). .. Total STAT5 levels were determined by staining with a purified pan-STAT5 antibody (89, BD Pharmingen) followed by a F(ab’)2 donkey anti-mouse secondary antibody conjugate (Jackson ImmunoResearch).

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    Becton Dickinson anti phosphorylated stat1
    TYK2 deficiency impairs the response to IL-23 but not to IL-27 nor IFN-γ. (A) Western blot analysis of <t>STAT1</t> (pSTAT1, top) and STAT3 (pSTAT3, bottom) phosphorylation in EBV–B cells from healthy controls (C1 and C2), TYK2-deficient patients (P1 and P2), a patient with complete STAT1 deficiency (STAT1*), and an AD-HIES patient with a heterozygous STAT3 mutation (WT/T708N; STAT3*), after stimulation with 100 ng/ml IL-27 for 20 min. STAT1, STAT3, and α-tubulin levels were also assessed. The results shown are representative of at least two independent experiments. (B) Microarray analysis of HVS–T cell lines from three healthy controls, P2, and an IL-12Rβ1–deficient patient. Cells were stimulated for 12 h with 100 ng/ml IL-23. The difference between nonstimulated and stimulated cultures is shown as a fold change. (C) Western blot depicting phospho-STAT3 (pSTAT3) in EBV–B cells from a healthy control (C, C1, and C2), TYK2-deficient patients (P1, P2, P3, P5, P7, and P8), an AD-HIES patient carrying a heterozygous STAT3 mutation (WT/T708N; STAT3*), an IL-12Rβ1–deficient patient (IL-12Rβ1*), and a STAT1-deficient patient (STAT1*), without (−) and with (+) stimulation for 30 min with 100 ng/ml IL-23. α-Tubulin was used as a protein loading control. The results shown are representative of at least three independent experiments. After analysis by densitometry, a p-value
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    STAT proteins in sperm subcellular fractions. A ) Following fractionation by nitrogen cavitation, sonication, and different centrifugation, sperm proteins were submitted to electrophoresis and transferred on PVDF membrane for immunoblotting with <t>anti-STAT1,</t>
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    TYK2 deficiency impairs the response to IL-23 but not to IL-27 nor IFN-γ. (A) Western blot analysis of STAT1 (pSTAT1, top) and STAT3 (pSTAT3, bottom) phosphorylation in EBV–B cells from healthy controls (C1 and C2), TYK2-deficient patients (P1 and P2), a patient with complete STAT1 deficiency (STAT1*), and an AD-HIES patient with a heterozygous STAT3 mutation (WT/T708N; STAT3*), after stimulation with 100 ng/ml IL-27 for 20 min. STAT1, STAT3, and α-tubulin levels were also assessed. The results shown are representative of at least two independent experiments. (B) Microarray analysis of HVS–T cell lines from three healthy controls, P2, and an IL-12Rβ1–deficient patient. Cells were stimulated for 12 h with 100 ng/ml IL-23. The difference between nonstimulated and stimulated cultures is shown as a fold change. (C) Western blot depicting phospho-STAT3 (pSTAT3) in EBV–B cells from a healthy control (C, C1, and C2), TYK2-deficient patients (P1, P2, P3, P5, P7, and P8), an AD-HIES patient carrying a heterozygous STAT3 mutation (WT/T708N; STAT3*), an IL-12Rβ1–deficient patient (IL-12Rβ1*), and a STAT1-deficient patient (STAT1*), without (−) and with (+) stimulation for 30 min with 100 ng/ml IL-23. α-Tubulin was used as a protein loading control. The results shown are representative of at least three independent experiments. After analysis by densitometry, a p-value

    Journal: The Journal of Experimental Medicine

    Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

    doi: 10.1084/jem.20140280

    Figure Lengend Snippet: TYK2 deficiency impairs the response to IL-23 but not to IL-27 nor IFN-γ. (A) Western blot analysis of STAT1 (pSTAT1, top) and STAT3 (pSTAT3, bottom) phosphorylation in EBV–B cells from healthy controls (C1 and C2), TYK2-deficient patients (P1 and P2), a patient with complete STAT1 deficiency (STAT1*), and an AD-HIES patient with a heterozygous STAT3 mutation (WT/T708N; STAT3*), after stimulation with 100 ng/ml IL-27 for 20 min. STAT1, STAT3, and α-tubulin levels were also assessed. The results shown are representative of at least two independent experiments. (B) Microarray analysis of HVS–T cell lines from three healthy controls, P2, and an IL-12Rβ1–deficient patient. Cells were stimulated for 12 h with 100 ng/ml IL-23. The difference between nonstimulated and stimulated cultures is shown as a fold change. (C) Western blot depicting phospho-STAT3 (pSTAT3) in EBV–B cells from a healthy control (C, C1, and C2), TYK2-deficient patients (P1, P2, P3, P5, P7, and P8), an AD-HIES patient carrying a heterozygous STAT3 mutation (WT/T708N; STAT3*), an IL-12Rβ1–deficient patient (IL-12Rβ1*), and a STAT1-deficient patient (STAT1*), without (−) and with (+) stimulation for 30 min with 100 ng/ml IL-23. α-Tubulin was used as a protein loading control. The results shown are representative of at least three independent experiments. After analysis by densitometry, a p-value

    Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

    Techniques: Western Blot, Mutagenesis, Microarray

    TYK2-deficient cells display an impaired response to IL-10 family cytokines. (A) The induction of IFIT1 transcription was analyzed by RT-qPCR after the treatment for 2 h with 20 ng/ml IL-29 or IL-28B of EBV–B cells from two healthy controls (C1 and C2), two TYK2-deficient patients (P1 and P2), a patient with complete STAT1 deficiency (STAT1*), and an IL-10R2–deficient patient (IL-10R2*). Results are normalized with respect to GUS levels. Error bars indicate SEM. (B) EMSA was performed with a GAS probe and EBV–B cells from a healthy control (C), TYK2-deficient patients (P1 and P2), and an IL-10R2–deficient patient (IL-10R2*), with (+) and without (−) stimulation with 50 ng/ml IL-10 for 20 min. (C) Western blot of EBV–B cells from a healthy control (C), TYK2-deficient patients (P1, P2, P3, P5, P7, and P8), and an IL-10R2–deficient patient (IL-10R2*) after treatment with 50 ng/ml IL-10 for 15 min, probed with an Ab specific for phosphorylated STAT3 (pSTAT3). An Ab against tubulin was used as a loading control. After analysis by densitometry, a nonsignificant p-value for the two-tailed Student’s t test for the comparison of IL-10–pSTAT3 controls and TYK2-deficient patients (TYK2) was found. (B and C) White lines indicate that intervening lanes were spliced out. (D) Western blot showing the detection of phospho-STAT3 (pSTAT3) in mock-transduced (left) or TYK2-transduced (right) EBV–B cells from a healthy control and P1, P2, P3, and P5, without (−) and with (+) stimulation for 30 min with 50 ng/ml IL-10. (E) SOCS3 induction was analyzed by RT-qPCR after 6 h of treatment with 50 ng/ml IL-10, in EBV–B cells from two healthy controls (C1 and C2), TYK2-deficient patients (P1 and P2), and an IL-10R2–deficient (IL-10R2*) patient. Results are normalized with respect to GUS . The results shown in A–E are representative of at least two independent experiments. (F) Impaired response to IL-10 in the macrophages of P2. The inhibition of TNF production in response to LPS and IL-10 was assessed by ELISA in macrophages from a travel control (C), P2’s relatives (mother, father, and sister), P2, and a patient with AD-HIES (STAT3*). (G and H) Western blots assessing the phosphorylation of STAT3 (pSTAT3) in healthy controls (C1 and C2), a TYK2-deficient patient (P2), an AD-HIES patient (STAT3*), and a patient with complete STAT1 deficiency (STAT1*), with (+) or without (−) LIF (100 ng/ml for 15 min). (G) In SV40-fibroblasts. (H) In primary fibroblasts. (I and J) Total CD4 and CD8 T cells (I) and naive (CCR7 + CD45RA + ), central memory (CCR7 + CD45RA − ; cmem), effector memory (CCR7 − CD45RA − ; emem), and revertant effector memory (CCR7 − CD45RA + ) T cells in CD4 + (J, left) and CD8 + T cells (J, right) from healthy controls (C) and the TYK2-deficient patients P1, P2 (tested three times), P4 (tested twice), and P5 (TYK2). Mean values for each set of conditions are indicated by solid lines.

    Journal: The Journal of Experimental Medicine

    Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

    doi: 10.1084/jem.20140280

    Figure Lengend Snippet: TYK2-deficient cells display an impaired response to IL-10 family cytokines. (A) The induction of IFIT1 transcription was analyzed by RT-qPCR after the treatment for 2 h with 20 ng/ml IL-29 or IL-28B of EBV–B cells from two healthy controls (C1 and C2), two TYK2-deficient patients (P1 and P2), a patient with complete STAT1 deficiency (STAT1*), and an IL-10R2–deficient patient (IL-10R2*). Results are normalized with respect to GUS levels. Error bars indicate SEM. (B) EMSA was performed with a GAS probe and EBV–B cells from a healthy control (C), TYK2-deficient patients (P1 and P2), and an IL-10R2–deficient patient (IL-10R2*), with (+) and without (−) stimulation with 50 ng/ml IL-10 for 20 min. (C) Western blot of EBV–B cells from a healthy control (C), TYK2-deficient patients (P1, P2, P3, P5, P7, and P8), and an IL-10R2–deficient patient (IL-10R2*) after treatment with 50 ng/ml IL-10 for 15 min, probed with an Ab specific for phosphorylated STAT3 (pSTAT3). An Ab against tubulin was used as a loading control. After analysis by densitometry, a nonsignificant p-value for the two-tailed Student’s t test for the comparison of IL-10–pSTAT3 controls and TYK2-deficient patients (TYK2) was found. (B and C) White lines indicate that intervening lanes were spliced out. (D) Western blot showing the detection of phospho-STAT3 (pSTAT3) in mock-transduced (left) or TYK2-transduced (right) EBV–B cells from a healthy control and P1, P2, P3, and P5, without (−) and with (+) stimulation for 30 min with 50 ng/ml IL-10. (E) SOCS3 induction was analyzed by RT-qPCR after 6 h of treatment with 50 ng/ml IL-10, in EBV–B cells from two healthy controls (C1 and C2), TYK2-deficient patients (P1 and P2), and an IL-10R2–deficient (IL-10R2*) patient. Results are normalized with respect to GUS . The results shown in A–E are representative of at least two independent experiments. (F) Impaired response to IL-10 in the macrophages of P2. The inhibition of TNF production in response to LPS and IL-10 was assessed by ELISA in macrophages from a travel control (C), P2’s relatives (mother, father, and sister), P2, and a patient with AD-HIES (STAT3*). (G and H) Western blots assessing the phosphorylation of STAT3 (pSTAT3) in healthy controls (C1 and C2), a TYK2-deficient patient (P2), an AD-HIES patient (STAT3*), and a patient with complete STAT1 deficiency (STAT1*), with (+) or without (−) LIF (100 ng/ml for 15 min). (G) In SV40-fibroblasts. (H) In primary fibroblasts. (I and J) Total CD4 and CD8 T cells (I) and naive (CCR7 + CD45RA + ), central memory (CCR7 + CD45RA − ; cmem), effector memory (CCR7 − CD45RA − ; emem), and revertant effector memory (CCR7 − CD45RA + ) T cells in CD4 + (J, left) and CD8 + T cells (J, right) from healthy controls (C) and the TYK2-deficient patients P1, P2 (tested three times), P4 (tested twice), and P5 (TYK2). Mean values for each set of conditions are indicated by solid lines.

    Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

    Techniques: Quantitative RT-PCR, Western Blot, Two Tailed Test, Inhibition, Enzyme-linked Immunosorbent Assay

    The response to type I IFNs in TYK2-deficient cells is impaired but not abolished. (A–D) Western blot of proteins extracted from EBV–B cells from a healthy control (C), TYK2-deficient patients (P1, P2, P5, P7, and P8), and a STAT1-deficient patient (STAT1*), with and without IFN-α (10 5 IU/ml) or IFN-β (3.2 × 10 4 IU/ml) stimulation for 30 min. (A and B) Abs against phosphorylated STAT1 (pSTAT1), STAT1, and tubulin were used. After analysis by densitometry, a p-value

    Journal: The Journal of Experimental Medicine

    Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

    doi: 10.1084/jem.20140280

    Figure Lengend Snippet: The response to type I IFNs in TYK2-deficient cells is impaired but not abolished. (A–D) Western blot of proteins extracted from EBV–B cells from a healthy control (C), TYK2-deficient patients (P1, P2, P5, P7, and P8), and a STAT1-deficient patient (STAT1*), with and without IFN-α (10 5 IU/ml) or IFN-β (3.2 × 10 4 IU/ml) stimulation for 30 min. (A and B) Abs against phosphorylated STAT1 (pSTAT1), STAT1, and tubulin were used. After analysis by densitometry, a p-value

    Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

    Techniques: Western Blot

    CMTR1 is required for the antiviral response. (A) Focus-forming assay of supernatants or (B) RT-qPCR analysis of viral RNA, relative to GAPDH , harvested from ZIKV- or DENV-infected Huh7 cells (48 hours post-infection (hpi); multiplicity of infection (MOI) 0.01) treated with the indicated siRNAs. (C) (Left) Representative fields of ZIKV- and DENV-infected Huh7 cells (48 hpi; MOI 0.01) treated with the indicated siRNAs and immunostained with anti-flaviviral E protein (green). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (Right) Quantification of the percentage of ZIKV- and DENV-infected Huh7 cells. (D) Quantification of the relative number of VSV-GFP-positive Huh7 WT and STAT1 KO cells after siRNA treatment and IFN-β pre-treatment (25 U/mL; 16 h) at 8 hpi and set to 100. For (C) and (D): ≥5000 cells were counted in each experiment per condition. Values are the mean ± SEM of 3 biological replicates. * p

    Journal: bioRxiv

    Article Title: The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes

    doi: 10.1101/2020.03.05.980045

    Figure Lengend Snippet: CMTR1 is required for the antiviral response. (A) Focus-forming assay of supernatants or (B) RT-qPCR analysis of viral RNA, relative to GAPDH , harvested from ZIKV- or DENV-infected Huh7 cells (48 hours post-infection (hpi); multiplicity of infection (MOI) 0.01) treated with the indicated siRNAs. (C) (Left) Representative fields of ZIKV- and DENV-infected Huh7 cells (48 hpi; MOI 0.01) treated with the indicated siRNAs and immunostained with anti-flaviviral E protein (green). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (Right) Quantification of the percentage of ZIKV- and DENV-infected Huh7 cells. (D) Quantification of the relative number of VSV-GFP-positive Huh7 WT and STAT1 KO cells after siRNA treatment and IFN-β pre-treatment (25 U/mL; 16 h) at 8 hpi and set to 100. For (C) and (D): ≥5000 cells were counted in each experiment per condition. Values are the mean ± SEM of 3 biological replicates. * p

    Article Snippet: The following antibodies were used for immunoblot analysis: rabbit anti-CMTR1 (Atlas Antibodies, HPA029980, 1:1000); mouse anti-ISG15 (Santa Cruz Biotechnology, sc-166755, 1:1000); mouse anti-IFITM1 (Proteintech, 60074-Ig, 1:1000); mouse anti-Tubulin (Sigma, T5168, 1:5000); rabbit anti-MX1 (Abcam, ab207414, 1:1000); mouse anti-IFIT3 (Abcam, ab76818, 1:1000); rabbit anti-IFIT1 (gift of Dr. Ganes Sen, ( )); mouse anti-pSTAT1 (pY701; BD, 612132, 1:1000); rabbit anti-GAPDH (GeneTex, GT239, 1:1000) mouse anti-STAT1 (BD, 610115, 1:1000); mouse anti-FLAG (Sigma, F3165, 1:5000).

    Techniques: Focus Forming Assay, Quantitative RT-PCR, Infection, Staining

    STAT proteins in sperm subcellular fractions. A ) Following fractionation by nitrogen cavitation, sonication, and different centrifugation, sperm proteins were submitted to electrophoresis and transferred on PVDF membrane for immunoblotting with anti-STAT1,

    Journal: Biology of Reproduction

    Article Title: Mediators of the JAK/STAT Signaling Pathway in Human Spermatozoa 1

    doi: 10.1095/biolreprod.111.092379

    Figure Lengend Snippet: STAT proteins in sperm subcellular fractions. A ) Following fractionation by nitrogen cavitation, sonication, and different centrifugation, sperm proteins were submitted to electrophoresis and transferred on PVDF membrane for immunoblotting with anti-STAT1,

    Article Snippet: The membrane was next incubated with an anti-IL6R polyclonal antibody (Santa Cruz Biotechnology), anti-JAK1, anti-STAT1, STAT3, STAT5, or STAT6 (BD Biosciences-Transduction Laboratories) monoclonal antibodies for 1 h at room temperature.

    Techniques: Fractionation, Sonication, Centrifugation, Electrophoresis

    Localization of IL6R, IL6ST, JAK1, STAT1, and STAT4 on sperm. Fixed and permeabilized spermatozoa were submitted to indirect immunofluorescence using an anti-JAK1 or anti-STAT1 monoclonal antibody ( D and E , respectively) or an anti-IL6R, anti-IL6ST, or

    Journal: Biology of Reproduction

    Article Title: Mediators of the JAK/STAT Signaling Pathway in Human Spermatozoa 1

    doi: 10.1095/biolreprod.111.092379

    Figure Lengend Snippet: Localization of IL6R, IL6ST, JAK1, STAT1, and STAT4 on sperm. Fixed and permeabilized spermatozoa were submitted to indirect immunofluorescence using an anti-JAK1 or anti-STAT1 monoclonal antibody ( D and E , respectively) or an anti-IL6R, anti-IL6ST, or

    Article Snippet: The membrane was next incubated with an anti-IL6R polyclonal antibody (Santa Cruz Biotechnology), anti-JAK1, anti-STAT1, STAT3, STAT5, or STAT6 (BD Biosciences-Transduction Laboratories) monoclonal antibodies for 1 h at room temperature.

    Techniques: Immunofluorescence

    Inhibition of interferon (IFN)-inducible protein-10 (CXCL10) release and signal transducer and activator of transcription (STAT1) phosphorylation by exogenous C3bi and/or C4b. (a) Purified C3bi and/or C4b was added to the whole blood culture assay. Blood

    Journal: Clinical and Experimental Immunology

    Article Title: Inhibition of CXCL10 release by monomeric C3bi and C4b

    doi: 10.1111/j.1365-2249.2011.04490.x

    Figure Lengend Snippet: Inhibition of interferon (IFN)-inducible protein-10 (CXCL10) release and signal transducer and activator of transcription (STAT1) phosphorylation by exogenous C3bi and/or C4b. (a) Purified C3bi and/or C4b was added to the whole blood culture assay. Blood

    Article Snippet: For the staining method that involved the use of an anti-STAT1 mAb, whole blood was treated by BD Phosflow Lyse/Fix buffer and BD Phosflow Perm buffer III (BD Biosciences) according to the BD Phosflow Protocols (Protocol III).

    Techniques: Inhibition, Purification