anti stat1 antibody  (Santa Cruz Biotechnology)

 
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    Name:
    p Stat1 Antibody
    Description:
    p Stat1 Antibody A 2 is a mouse monoclonal IgG1 kappa light chain provided at 200 µg ml raised against Stat1 of human origin Anti p Stat1 Antibody A 2 is recommended for detection of Stat1 phosphorylated at Tyr 701 of mouse rat and human origin by WB IP IF FCM and ELISA Anti p Stat1 Antibody A 2 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to either PerCP or PerCP Cy5 5 for IF IHC P and FCM TransCruz reagent for Gel Supershift and ChIP applications sc 8394 X 200 µg 0 1 ml blocking peptide sc 8394 P Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of p Stat1 A 2 sc 8394
    Catalog Number:
    SC-8394
    Price:
    None
    Category:
    Antibodies Primary Antibodies and ImmunoCruz Conjugates Transcription Regulators Stat1 Antibodies p Stat1 Antibody A 2
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    Structured Review

    Santa Cruz Biotechnology anti stat1 antibody
    <t>STAT1</t> binds to T FH cell signature genes
    p Stat1 Antibody A 2 is a mouse monoclonal IgG1 kappa light chain provided at 200 µg ml raised against Stat1 of human origin Anti p Stat1 Antibody A 2 is recommended for detection of Stat1 phosphorylated at Tyr 701 of mouse rat and human origin by WB IP IF FCM and ELISA Anti p Stat1 Antibody A 2 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to either PerCP or PerCP Cy5 5 for IF IHC P and FCM TransCruz reagent for Gel Supershift and ChIP applications sc 8394 X 200 µg 0 1 ml blocking peptide sc 8394 P Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of p Stat1 A 2 sc 8394
    https://www.bioz.com/result/anti stat1 antibody/product/Santa Cruz Biotechnology
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti stat1 antibody - by Bioz Stars, 2021-05
    94/100 stars

    Images

    1) Product Images from "Type I Interferon induces binding of STAT1 to Bcl6: Divergent Roles of STAT-family transcription factors in the TFH cell genetic program"

    Article Title: Type I Interferon induces binding of STAT1 to Bcl6: Divergent Roles of STAT-family transcription factors in the TFH cell genetic program

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

    doi: 10.4049/jimmunol.1300675

    STAT1 binds to T FH cell signature genes
    Figure Legend Snippet: STAT1 binds to T FH cell signature genes

    Techniques Used:

    STAT1, but not STAT3 or STAT4 is required for type I IFN mediated induction of key T FH cell molecules
    Figure Legend Snippet: STAT1, but not STAT3 or STAT4 is required for type I IFN mediated induction of key T FH cell molecules

    Techniques Used:

    2) Product Images from "Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway"

    Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-51998-w

    Our proposed model of nsDNA-induced APOL1 expression through engagement of the cGAS/IFI16-STING pathway in human immortalized AB8/13 podocytes. Binding of cytosolic nsDNA by cGAS and IFI16 activates STING, which subsequently activates TBK1. Activated TBK1 phosphorylates IRF3, which promotes transcription of APOL1 and IFNβ . IFNβ released from the cells (or exogenous IFNβ) binds to IFNAR. IFNAR-associated JAK1 and Tyk2 kinases then phosphorylate STAT1, which promotes transcription of APOL1 and IFI16 . A putative IFI16-mediated activation of STING is indicated by a dashed arrow. A potential cooperation between cGAS and IFI16 is indicated by a double-headed arrow. Deficient STING phosphorylation observed in cGAS- or IFI16-knockdown cells (Fig. 6 ) suggests that nsDNA-induced APOL1 expression may be mediated by a phospho-STING-independent pathway, marked by a green arrow. A dual JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) suppresses STAT1 activation and thereby inhibits IFI16 expression and STAT1-mediated APOL1 expression. Since IFNAR-mediated signaling involves JAK1 but not JAK2, our model only depicts the inhibition of JAK1 by Ruxo. Ruxo does not affect IRF3-mediated APOL1 expression.
    Figure Legend Snippet: Our proposed model of nsDNA-induced APOL1 expression through engagement of the cGAS/IFI16-STING pathway in human immortalized AB8/13 podocytes. Binding of cytosolic nsDNA by cGAS and IFI16 activates STING, which subsequently activates TBK1. Activated TBK1 phosphorylates IRF3, which promotes transcription of APOL1 and IFNβ . IFNβ released from the cells (or exogenous IFNβ) binds to IFNAR. IFNAR-associated JAK1 and Tyk2 kinases then phosphorylate STAT1, which promotes transcription of APOL1 and IFI16 . A putative IFI16-mediated activation of STING is indicated by a dashed arrow. A potential cooperation between cGAS and IFI16 is indicated by a double-headed arrow. Deficient STING phosphorylation observed in cGAS- or IFI16-knockdown cells (Fig. 6 ) suggests that nsDNA-induced APOL1 expression may be mediated by a phospho-STING-independent pathway, marked by a green arrow. A dual JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) suppresses STAT1 activation and thereby inhibits IFI16 expression and STAT1-mediated APOL1 expression. Since IFNAR-mediated signaling involves JAK1 but not JAK2, our model only depicts the inhibition of JAK1 by Ruxo. Ruxo does not affect IRF3-mediated APOL1 expression.

    Techniques Used: Expressing, Binding Assay, Activation Assay, Inhibition

    nsDNA-induced APOL1 expression is partly attenuated by JAK1/JAK2 inhibitor Ruxolitinib. ( a ) Ruxolitinib (Ruxo) inhibited IFNβ-mediated phosphorylation of STAT1. Sets of AB8/13 podocytes were treated with DMSO (solvent) only (Control), treated for 2 h with 5 μM Ruxo, treated for 15 min with 10 ng ml −1 IFNβ, or pretreated with Ruxo for 2 h followed by IFNβ stimulation for 15 min (Ruxo/IFNβ). The blot images were obtained from different gels. The blot probed for P-STAT1 was re-probed for GAPDH. ( b , c ) AB8/13 podocytes were treated with Ruxo and IFNβ as indicated and subsequently transfected with 1 μg ml −1 nsDNA for 2 h ( b ) or 18 h ( c ). The IFI16/GAPDH and APOL1/GAPDH ratios in unstimulated cells (Control) were both set as 1.0. The blot images in ( b ) were obtained from different gels. The blot probed for P-IRF3 was re-probed for P-STING. Other blot images were cropped from individually probed blots. The blot images in ( c ) were obtained from different gels. The blot probed for IFI16 was re-probed for cGAS. Other blot images were cropped from individually probed blots. Full images of all blots are shown in Supplementary Fig. S7 . ( d , e ) Ruxo abolished expression of APOL1 ( d ) and IFI16 mRNA ( e ) induced by exogenous IFNβ. ( f , g ) Ruxo partially inhibited nsDNA-induced APOL1 mRNA expression ( f ) but abolished nsDNA-induced IFI16 mRNA expression ( g ). Expression of APOL1 and IFNβ mRNA was analyzed by qRT-PCR 18 h after transfection with 1 μg ml −1 nsDNA. mRNA expression was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).
    Figure Legend Snippet: nsDNA-induced APOL1 expression is partly attenuated by JAK1/JAK2 inhibitor Ruxolitinib. ( a ) Ruxolitinib (Ruxo) inhibited IFNβ-mediated phosphorylation of STAT1. Sets of AB8/13 podocytes were treated with DMSO (solvent) only (Control), treated for 2 h with 5 μM Ruxo, treated for 15 min with 10 ng ml −1 IFNβ, or pretreated with Ruxo for 2 h followed by IFNβ stimulation for 15 min (Ruxo/IFNβ). The blot images were obtained from different gels. The blot probed for P-STAT1 was re-probed for GAPDH. ( b , c ) AB8/13 podocytes were treated with Ruxo and IFNβ as indicated and subsequently transfected with 1 μg ml −1 nsDNA for 2 h ( b ) or 18 h ( c ). The IFI16/GAPDH and APOL1/GAPDH ratios in unstimulated cells (Control) were both set as 1.0. The blot images in ( b ) were obtained from different gels. The blot probed for P-IRF3 was re-probed for P-STING. Other blot images were cropped from individually probed blots. The blot images in ( c ) were obtained from different gels. The blot probed for IFI16 was re-probed for cGAS. Other blot images were cropped from individually probed blots. Full images of all blots are shown in Supplementary Fig. S7 . ( d , e ) Ruxo abolished expression of APOL1 ( d ) and IFI16 mRNA ( e ) induced by exogenous IFNβ. ( f , g ) Ruxo partially inhibited nsDNA-induced APOL1 mRNA expression ( f ) but abolished nsDNA-induced IFI16 mRNA expression ( g ). Expression of APOL1 and IFNβ mRNA was analyzed by qRT-PCR 18 h after transfection with 1 μg ml −1 nsDNA. mRNA expression was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    3) Product Images from "The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARα/STAT1 axis"

    Article Title: The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARα/STAT1 axis

    Journal: Scientific Reports

    doi: 10.1038/srep24589

    Effect of TAK165 and ATRA on STAT1 activation. ( a ) Western blot analysis of p-STAT1 (Tyr701 and Ser727) and STAT1 in the HL60 and NB4 cells. The cells were treated with TAK165 (100 nM for HL60, 50 nM for NB4) and ATRA (1 μM for HL60, 2 nM for NB4) for 3 days. ( b ) The relative phosphorylation level of STAT1 at Tyr701 and Ser727 in the HL60 cells, were evaluated by p-STAT1 (Tyr701 or Ser727)/STAT1. ( c ) STAT1 expression in the NB4-scrambleand NB4-shRNA-STAT1 cells was measured by western blot. ( d ) CD11b expression in the NB4-scrambleand NB4-shRNA-STAT1 cells. The cells were treated with 50 nM TAK165 and 2 nM ATRA for 3 days. The data presented are the mean ± SD. *** p
    Figure Legend Snippet: Effect of TAK165 and ATRA on STAT1 activation. ( a ) Western blot analysis of p-STAT1 (Tyr701 and Ser727) and STAT1 in the HL60 and NB4 cells. The cells were treated with TAK165 (100 nM for HL60, 50 nM for NB4) and ATRA (1 μM for HL60, 2 nM for NB4) for 3 days. ( b ) The relative phosphorylation level of STAT1 at Tyr701 and Ser727 in the HL60 cells, were evaluated by p-STAT1 (Tyr701 or Ser727)/STAT1. ( c ) STAT1 expression in the NB4-scrambleand NB4-shRNA-STAT1 cells was measured by western blot. ( d ) CD11b expression in the NB4-scrambleand NB4-shRNA-STAT1 cells. The cells were treated with 50 nM TAK165 and 2 nM ATRA for 3 days. The data presented are the mean ± SD. *** p

    Techniques Used: Activation Assay, Western Blot, Expressing, shRNA

    4) Product Images from "PHOSPHORYLATION OF Y372 IS CRITICAL FOR JAK2 TYROSINE KINASE ACTIVATION"

    Article Title: PHOSPHORYLATION OF Y372 IS CRITICAL FOR JAK2 TYROSINE KINASE ACTIVATION

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2011.06.015

    Loss of Y372 phosphorylation reduces Jak2-mediated STAT1 phosphorylation
    Figure Legend Snippet: Loss of Y372 phosphorylation reduces Jak2-mediated STAT1 phosphorylation

    Techniques Used:

    The Jak2-Y372F mutant has an impaired ability to co-associate with STAT1
    Figure Legend Snippet: The Jak2-Y372F mutant has an impaired ability to co-associate with STAT1

    Techniques Used: Mutagenesis

    5) Product Images from "Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation"

    Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

    Journal: Journal of Virology

    doi: 10.1128/JVI.01136-17

    Influence of NiV-N on the STAT nuclear import system. (A) The associations between STAT1 and Impα5, Impα6, and Impα7 in the presence of N protein were evaluated by immunoprecipitation (IP). The myc-tagged importins were precipitated with an anti-myc antibody, and the coprecipitation of pSTAT1 with Impα5, Impα6, or Impα7 was evaluated in the presence or absence of N protein. A myc-tagged Impα1 construct was employed as a negative control. P protein served as a positive-control antagonist of the interaction between Impα5 and STAT1. (B) The interaction between NiV-N and HA-Impβ1 or HA-Ran was investigated with immunoprecipitation assays using anti-HA and anti-NiV-N antibodies. The myc-Impα5, myc-Impβ1, and NiV-P proteins were included as positive controls for immunoprecipitation. (C) 293T cells were transfected with pCAGGS-NiV-N (encoding a C-terminal HA tag). After 24 h, the cells were treated with 1,000 U/ml IFN-α for 30 min, and immunoprecipitation was conducted using anti-HA antibody. STAT1, STAT2, STAT3, and N protein in the lysates were detected with anti-STAT1 (E-23), -2 (C-20), and -3 (H-190) and anti-N protein antibodies, respectively. The P protein served as a positive control for an N-binding protein. For panels A to C, the experiments were independently repeated three times, and representative blots are displayed. (D) A reporter assay was conducted using 293T cells transfected with the NiV-N or N-S451A plasmid. The expression levels of each N protein and GAPDH are shown. Error bars indicate standard deviations. n.s., not significant. N-S451A-expressing Cos7 cells were treated with 2,000 U/ml of IFN-α. N-S451A and STAT1 were detected with specific antibodies and are shown as z-stack immunofluorescence images. Arrowheads and arrows indicate an N-S451A-expressing and a non-N-S451A-expressing cell, respectively. The experiment was independently conducted three times. (E) The expression plasmids for NiV-N and EGFP-Kir/Gem-W268G (referred to here as rKir/Gem) were transfected into Cos7 cells, and NiV-N was detected with an anti-NiV-N polyclonal antibody. The nuclei were stained with Hoechst dye. Images shown are z-stack data. The arrowheads and arrows point to a NiV-N-expressing and a non-NiV-N-expressing cell, respectively. The bar graph indicates the statistical evaluation of the rKir/Gem distribution. The scores were determined by counting approximately 60 cells from five randomly selected fields. n.s., not significant.
    Figure Legend Snippet: Influence of NiV-N on the STAT nuclear import system. (A) The associations between STAT1 and Impα5, Impα6, and Impα7 in the presence of N protein were evaluated by immunoprecipitation (IP). The myc-tagged importins were precipitated with an anti-myc antibody, and the coprecipitation of pSTAT1 with Impα5, Impα6, or Impα7 was evaluated in the presence or absence of N protein. A myc-tagged Impα1 construct was employed as a negative control. P protein served as a positive-control antagonist of the interaction between Impα5 and STAT1. (B) The interaction between NiV-N and HA-Impβ1 or HA-Ran was investigated with immunoprecipitation assays using anti-HA and anti-NiV-N antibodies. The myc-Impα5, myc-Impβ1, and NiV-P proteins were included as positive controls for immunoprecipitation. (C) 293T cells were transfected with pCAGGS-NiV-N (encoding a C-terminal HA tag). After 24 h, the cells were treated with 1,000 U/ml IFN-α for 30 min, and immunoprecipitation was conducted using anti-HA antibody. STAT1, STAT2, STAT3, and N protein in the lysates were detected with anti-STAT1 (E-23), -2 (C-20), and -3 (H-190) and anti-N protein antibodies, respectively. The P protein served as a positive control for an N-binding protein. For panels A to C, the experiments were independently repeated three times, and representative blots are displayed. (D) A reporter assay was conducted using 293T cells transfected with the NiV-N or N-S451A plasmid. The expression levels of each N protein and GAPDH are shown. Error bars indicate standard deviations. n.s., not significant. N-S451A-expressing Cos7 cells were treated with 2,000 U/ml of IFN-α. N-S451A and STAT1 were detected with specific antibodies and are shown as z-stack immunofluorescence images. Arrowheads and arrows indicate an N-S451A-expressing and a non-N-S451A-expressing cell, respectively. The experiment was independently conducted three times. (E) The expression plasmids for NiV-N and EGFP-Kir/Gem-W268G (referred to here as rKir/Gem) were transfected into Cos7 cells, and NiV-N was detected with an anti-NiV-N polyclonal antibody. The nuclei were stained with Hoechst dye. Images shown are z-stack data. The arrowheads and arrows point to a NiV-N-expressing and a non-NiV-N-expressing cell, respectively. The bar graph indicates the statistical evaluation of the rKir/Gem distribution. The scores were determined by counting approximately 60 cells from five randomly selected fields. n.s., not significant.

    Techniques Used: Immunoprecipitation, Construct, Negative Control, Positive Control, Transfection, Binding Assay, Reporter Assay, Plasmid Preparation, Expressing, Immunofluorescence, Staining

    Inhibition of nuclear accumulation of STAT1 and -2 in the presence of NiV-N protein. (A) Cos7 or HeLa cells were transfected with 0.6 μg of the pCAGGS-NiV-N or -NiV-N-HA expression plasmid and were treated with IFN-α or -γ for 30 min. The cells were stained using anti-STAT1, -pSTAT1, -STAT2, and -pSTAT2 antibodies with an anti-N polyclonal antibody (or anti-HA mouse antibody) and Hoechst 33342 dye. The images shown are all z-stack data. Arrowheads indicate the cytoplasmic STATs in the NiV-N-expressing cells. The bar graphs show the statistical evaluation of STAT nuclear accumulation. We compared the scores for STAT distribution between NiV-N-expressing and non-NiV-N-expressing cells. The scores were calculated by counting approximately 60 cells from at least five randomly selected fields. Filled bars indicate the percentage of nuclear localization, while white bars indicate the percentage of whole-cell distribution. Error bars indicate standard deviations. The experiments were repeated three times independently. **, P
    Figure Legend Snippet: Inhibition of nuclear accumulation of STAT1 and -2 in the presence of NiV-N protein. (A) Cos7 or HeLa cells were transfected with 0.6 μg of the pCAGGS-NiV-N or -NiV-N-HA expression plasmid and were treated with IFN-α or -γ for 30 min. The cells were stained using anti-STAT1, -pSTAT1, -STAT2, and -pSTAT2 antibodies with an anti-N polyclonal antibody (or anti-HA mouse antibody) and Hoechst 33342 dye. The images shown are all z-stack data. Arrowheads indicate the cytoplasmic STATs in the NiV-N-expressing cells. The bar graphs show the statistical evaluation of STAT nuclear accumulation. We compared the scores for STAT distribution between NiV-N-expressing and non-NiV-N-expressing cells. The scores were calculated by counting approximately 60 cells from at least five randomly selected fields. Filled bars indicate the percentage of nuclear localization, while white bars indicate the percentage of whole-cell distribution. Error bars indicate standard deviations. The experiments were repeated three times independently. **, P

    Techniques Used: Inhibition, Transfection, Expressing, Plasmid Preparation, Staining

    Influence of N protein on expression of ISGs. (A) 293T cells were treated with 1,000 U/ml IFN-α in the presence or absence of NiV-N protein. The component proteins were fractionated into nuclear and cytoplasmic fractions. The whole-cell lysate and fractionated samples were subjected to SDS-PAGE to examine if the NiV-N protein decreased the nuclear pSTAT1 level. The relative nuclear STAT1 level (pSTAT1/histone H3) was measured by densitometric analysis using ImageJ software, and mean values for three independent experiments are shown. Error bars indicate standard deviations. (B) 293T cells were transfected with a NiV-N expression plasmid or empty vector, and a chromatin immunoprecipitation assay was carried out with anti-STAT1 antibody (E-23) or control IgG (ab37415-5) after treatment with 1,000 U/ml IFN-α. Enrichment of ISG promoters (IFIT2, MX2, OAS1, and PKR) was measured by quantitative PCR, and the amount of precipitated DNA relative to the amount of input DNA is shown as a percentage of the input. The experiment was repeated three times independently, and error bars show standard deviations. *, P
    Figure Legend Snippet: Influence of N protein on expression of ISGs. (A) 293T cells were treated with 1,000 U/ml IFN-α in the presence or absence of NiV-N protein. The component proteins were fractionated into nuclear and cytoplasmic fractions. The whole-cell lysate and fractionated samples were subjected to SDS-PAGE to examine if the NiV-N protein decreased the nuclear pSTAT1 level. The relative nuclear STAT1 level (pSTAT1/histone H3) was measured by densitometric analysis using ImageJ software, and mean values for three independent experiments are shown. Error bars indicate standard deviations. (B) 293T cells were transfected with a NiV-N expression plasmid or empty vector, and a chromatin immunoprecipitation assay was carried out with anti-STAT1 antibody (E-23) or control IgG (ab37415-5) after treatment with 1,000 U/ml IFN-α. Enrichment of ISG promoters (IFIT2, MX2, OAS1, and PKR) was measured by quantitative PCR, and the amount of precipitated DNA relative to the amount of input DNA is shown as a percentage of the input. The experiment was repeated three times independently, and error bars show standard deviations. *, P

    Techniques Used: Expressing, SDS Page, Software, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    6) Product Images from "IL-27 alleviates the bleomycin-induced pulmonary fibrosis by regulating the Th17 cell differentiation"

    Article Title: IL-27 alleviates the bleomycin-induced pulmonary fibrosis by regulating the Th17 cell differentiation

    Journal: BMC Pulmonary Medicine

    doi: 10.1186/s12890-015-0012-4

    The expression of JAK/STAT and TGF-β/Smad signaling pathway-related proteins in the lung tissues of the different groups on days 7 and 28. A . Western blots of JAK/STAT signaling pathway-related proteins: JAK, STAT1, STAT3, STAT5, SOCS3, and phosphorylated STAT1, STAT3, and STAT5. B . A histogram of the relative gray values shown in A . C . Western blots for the TGF-β/Smad signaling pathway-related proteins: Smad1, Smad3, TGF-βR1 and phosphorylated Smad1 and Smad3. D. A histogram of the relative gray values shown in C . Data are expressed as means ± SEM (n = 3). *p
    Figure Legend Snippet: The expression of JAK/STAT and TGF-β/Smad signaling pathway-related proteins in the lung tissues of the different groups on days 7 and 28. A . Western blots of JAK/STAT signaling pathway-related proteins: JAK, STAT1, STAT3, STAT5, SOCS3, and phosphorylated STAT1, STAT3, and STAT5. B . A histogram of the relative gray values shown in A . C . Western blots for the TGF-β/Smad signaling pathway-related proteins: Smad1, Smad3, TGF-βR1 and phosphorylated Smad1 and Smad3. D. A histogram of the relative gray values shown in C . Data are expressed as means ± SEM (n = 3). *p

    Techniques Used: Expressing, Western Blot

    7) Product Images from "Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages"

    Article Title: Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00709

    rM180 downregulates the IFNγ-induced expression of CIITA molecules in THP-1 cells. THP-1 cells were incubated with rM180 for 24 h, washed, and serum-starved for 24 h. After stimulation by IFNγ for the indicated times, whole cell lysates prepared from THP-1 cells were immunoblotted with various antibodies. β-actin was used as a control. Protein expression was quantified using the ImageJ program. Representative western blots of phosphorylated (A) JAK2 and (B) STAT1, and total (C) IFN regulatory factor-1 (IRF-1) and (D) class II transactivator (CIITA). Quantification of phosphorylated protein levels relative to total protein and total IRF-1 and CIITA protein levels relative to β-actin was carried out using ImageJ. The significance of differences between groups was determined by a two-tailed unpaired Student's test; ** P
    Figure Legend Snippet: rM180 downregulates the IFNγ-induced expression of CIITA molecules in THP-1 cells. THP-1 cells were incubated with rM180 for 24 h, washed, and serum-starved for 24 h. After stimulation by IFNγ for the indicated times, whole cell lysates prepared from THP-1 cells were immunoblotted with various antibodies. β-actin was used as a control. Protein expression was quantified using the ImageJ program. Representative western blots of phosphorylated (A) JAK2 and (B) STAT1, and total (C) IFN regulatory factor-1 (IRF-1) and (D) class II transactivator (CIITA). Quantification of phosphorylated protein levels relative to total protein and total IRF-1 and CIITA protein levels relative to β-actin was carried out using ImageJ. The significance of differences between groups was determined by a two-tailed unpaired Student's test; ** P

    Techniques Used: Expressing, Incubation, Western Blot, Two Tailed Test

    8) Product Images from "HO-1 Overexpressed Mesenchymal Stem Cells Ameliorate Sepsis-Associated Acute Kidney Injury by Activating JAK/stat3 Pathway"

    Article Title: HO-1 Overexpressed Mesenchymal Stem Cells Ameliorate Sepsis-Associated Acute Kidney Injury by Activating JAK/stat3 Pathway

    Journal: Cellular and Molecular Bioengineering

    doi: 10.1007/s12195-018-0540-0

    HO-1 MSCs activates JAK/stat3 signaling pathway 48 h after MSC treatment. (a) Western blot image of key proteins in the JAK/stat3 signaling pathway, including JAK1, P-JAK2, JAK2, P-STAT3, STAT3. The expression of β -actin was used as a loading control. (b)–(d) Quantitative comparison of the expression levels of P-JAK1, P-JAK2, and P-STAT3 between different groups. (e) Western blot image of STAT1 and P-STAT1. p value: Student’s t test and ANOVA. Error bar: SEM.
    Figure Legend Snippet: HO-1 MSCs activates JAK/stat3 signaling pathway 48 h after MSC treatment. (a) Western blot image of key proteins in the JAK/stat3 signaling pathway, including JAK1, P-JAK2, JAK2, P-STAT3, STAT3. The expression of β -actin was used as a loading control. (b)–(d) Quantitative comparison of the expression levels of P-JAK1, P-JAK2, and P-STAT3 between different groups. (e) Western blot image of STAT1 and P-STAT1. p value: Student’s t test and ANOVA. Error bar: SEM.

    Techniques Used: Western Blot, Expressing

    9) Product Images from "Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses"

    Article Title: Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses

    Journal: mBio

    doi: 10.1128/mBio.01174-14

    STAT1 binding corresponds with differential ISG expression. (A) RNA expression of consensus ISGs linked to STAT1 following H5N1-VN1203 infection. Values represent log 2 FC relative to time-matched mocks. (B) Chromatin immunoprecipitation with antibodies against phospho-STAT1 (Santa Cruz) followed by qPCR of the 5′UTR of upregulated genes CXCL10 and IFIT1 or downregulated genes CFHR1 and APOL6 in the context of H5N1-VN1203 infection. Values represent fold increase binding compared to mock on a linear scale.
    Figure Legend Snippet: STAT1 binding corresponds with differential ISG expression. (A) RNA expression of consensus ISGs linked to STAT1 following H5N1-VN1203 infection. Values represent log 2 FC relative to time-matched mocks. (B) Chromatin immunoprecipitation with antibodies against phospho-STAT1 (Santa Cruz) followed by qPCR of the 5′UTR of upregulated genes CXCL10 and IFIT1 or downregulated genes CFHR1 and APOL6 in the context of H5N1-VN1203 infection. Values represent fold increase binding compared to mock on a linear scale.

    Techniques Used: Binding Assay, Expressing, RNA Expression, Infection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    10) Product Images from "Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis"

    Article Title: Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.0513250

    Heterozygous mutations affecting the CCD and DBD of STAT1 were identified in two sporadic and four multiplex cases in 10 CMCD patients from Japan.
    Figure Legend Snippet: Heterozygous mutations affecting the CCD and DBD of STAT1 were identified in two sporadic and four multiplex cases in 10 CMCD patients from Japan.

    Techniques Used: Multiplex Assay

    Functional analysis of STAT1-mutated alleles using transient gene expression systems.
    Figure Legend Snippet: Functional analysis of STAT1-mutated alleles using transient gene expression systems.

    Techniques Used: Functional Assay, Expressing

    11) Product Images from "Secretome of Hypoxic Endothelial Cells Stimulates Bone Marrow-Derived Mesenchymal Stem Cells to Enhance Alternative Activation of Macrophages"

    Article Title: Secretome of Hypoxic Endothelial Cells Stimulates Bone Marrow-Derived Mesenchymal Stem Cells to Enhance Alternative Activation of Macrophages

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21124409

    Hypoxic endothelial cells activate the MSCs intracellular signaling pathways of IFN-γ and TNF-α. The culture media obtained from endothelial cells subjected to hypoxia for 4 h were used for culturing HCELL-positive MSCs in the presence of the inhibitors of signaling pathways of IFN-γ ( a ) and TNF-α (b), whereas those from intact endothelial cells were used for assessing recombinant IFN-γ ( a ) and TNF-α ( b ) stimulation. After 24 hours’ culture in the depicted experimental settings, MSCs pellets were subjected to Western blotting to measure p-STAT1 and NF-κB for assessing IFN-γ and TNF-α pathway activation respectively, while the supernatant was used to measure IL-13 level with ELISA. The representative Western blottings are shown. The quantitative results of Western blotting were adjusted according to β-actin level. n = 8 in each group; a, p
    Figure Legend Snippet: Hypoxic endothelial cells activate the MSCs intracellular signaling pathways of IFN-γ and TNF-α. The culture media obtained from endothelial cells subjected to hypoxia for 4 h were used for culturing HCELL-positive MSCs in the presence of the inhibitors of signaling pathways of IFN-γ ( a ) and TNF-α (b), whereas those from intact endothelial cells were used for assessing recombinant IFN-γ ( a ) and TNF-α ( b ) stimulation. After 24 hours’ culture in the depicted experimental settings, MSCs pellets were subjected to Western blotting to measure p-STAT1 and NF-κB for assessing IFN-γ and TNF-α pathway activation respectively, while the supernatant was used to measure IL-13 level with ELISA. The representative Western blottings are shown. The quantitative results of Western blotting were adjusted according to β-actin level. n = 8 in each group; a, p

    Techniques Used: Recombinant, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Bilobalide Alleviated Dextran Sulfate Sodium-Induced Experimental Colitis by Inhibiting M1 Macrophage Polarization Through the NF-κB Signaling Pathway"

    Article Title: Bilobalide Alleviated Dextran Sulfate Sodium-Induced Experimental Colitis by Inhibiting M1 Macrophage Polarization Through the NF-κB Signaling Pathway

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2020.00718

    Bilobalide inhibited NF-κB signaling which hampered differentiation of M1 macrophage. (A) Bone marrow-derived macrophages (BMDMs) were treated with 1, 3, and 10 μM bilobalide in the presence of 10 ng/ml lipopolysaccharide (LPS) and 10 ng/ml interferon-gamma (IFN-γ) (M1) for 30 min. The expression of MyD88, TRAF6, p-STAT1, STAT1, p-p65, and p65 were determined by western blot. (B) BMDMs were treated with 10 μM bilobalide in the presence of 10 ng/ml LPS and 10 ng/ml IFN-γ (M1) for 30 min. The cytosol and nuclei fraction were isolated and the level of p65 was determined by western blot. (C) The BMDMs were cultured as described above and stained with p65. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 10 μm.
    Figure Legend Snippet: Bilobalide inhibited NF-κB signaling which hampered differentiation of M1 macrophage. (A) Bone marrow-derived macrophages (BMDMs) were treated with 1, 3, and 10 μM bilobalide in the presence of 10 ng/ml lipopolysaccharide (LPS) and 10 ng/ml interferon-gamma (IFN-γ) (M1) for 30 min. The expression of MyD88, TRAF6, p-STAT1, STAT1, p-p65, and p65 were determined by western blot. (B) BMDMs were treated with 10 μM bilobalide in the presence of 10 ng/ml LPS and 10 ng/ml IFN-γ (M1) for 30 min. The cytosol and nuclei fraction were isolated and the level of p65 was determined by western blot. (C) The BMDMs were cultured as described above and stained with p65. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 10 μm.

    Techniques Used: Derivative Assay, Expressing, Western Blot, Isolation, Cell Culture, Staining

    13) Product Images from "Identification of 5-Methoxy-2-(Diformylmethylidene)-3,3-Dimethylindole as an Anti-Influenza A Virus Agent"

    Article Title: Identification of 5-Methoxy-2-(Diformylmethylidene)-3,3-Dimethylindole as an Anti-Influenza A Virus Agent

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170352

    526A inhibits IAV-induced IRF3 and STAT1 activation. The A549-PB1 cells were treated with various concentrations of 526A as indicated for four hours followed by PR8-PB1flank-eGFP IAV infection at an MOI of 1.0 for twelve hours. Whole cell extracts were prepared and immunoblotted with antibodies against PARP, p-IRF3 and p-STAT1.
    Figure Legend Snippet: 526A inhibits IAV-induced IRF3 and STAT1 activation. The A549-PB1 cells were treated with various concentrations of 526A as indicated for four hours followed by PR8-PB1flank-eGFP IAV infection at an MOI of 1.0 for twelve hours. Whole cell extracts were prepared and immunoblotted with antibodies against PARP, p-IRF3 and p-STAT1.

    Techniques Used: Activation Assay, Infection

    14) Product Images from "Experimental Colitis Is Associated with Transcriptional Inhibition of Na+/Ca2+ Exchanger Isoform 1 (NCX1) Expression by Interferon γ in the Renal Distal Convoluted Tubules *"

    Article Title: Experimental Colitis Is Associated with Transcriptional Inhibition of Na+/Ca2+ Exchanger Isoform 1 (NCX1) Expression by Interferon γ in the Renal Distal Convoluted Tubules *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.616516

    STAT1 is required to down-regulate the expression of NCX1 by IFNγ. WT and Stat1 −/− mice (129S6/SvEv background) were injected with PBS or mIFN γ through i.p for 3 days and kidneys were collected for RNA and protein extraction.
    Figure Legend Snippet: STAT1 is required to down-regulate the expression of NCX1 by IFNγ. WT and Stat1 −/− mice (129S6/SvEv background) were injected with PBS or mIFN γ through i.p for 3 days and kidneys were collected for RNA and protein extraction.

    Techniques Used: Expressing, Mouse Assay, Injection, Protein Extraction

    15) Product Images from "Role of STAT1 and Oxidative Stress in Gentamicin-Induced Hair Cell Death in Organ of Corti"

    Article Title: Role of STAT1 and Oxidative Stress in Gentamicin-Induced Hair Cell Death in Organ of Corti

    Journal: Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology

    doi: 10.1097/MAO.0000000000001192

    Prevalence of phosphorlyated STAT1 at Ser 727 is increased after 72 hours of 4 μM gentamicin therapy, and this increase is attenuated by EGCG
    Figure Legend Snippet: Prevalence of phosphorlyated STAT1 at Ser 727 is increased after 72 hours of 4 μM gentamicin therapy, and this increase is attenuated by EGCG

    Techniques Used:

    Representative morphology from basal turn demonstrating increased immunostaining for STAT1 Ser 727 phosphorlyation after gentamicin therapy
    Figure Legend Snippet: Representative morphology from basal turn demonstrating increased immunostaining for STAT1 Ser 727 phosphorlyation after gentamicin therapy

    Techniques Used: Immunostaining

    Retinoic acid, a STAT1 activator, potentiated gentamicin's toxicity in explant model
    Figure Legend Snippet: Retinoic acid, a STAT1 activator, potentiated gentamicin's toxicity in explant model

    Techniques Used:

    16) Product Images from "IFN-Dependent and -Independent Reduction in West Nile Virus Infectivity in Human Dermal Fibroblasts"

    Article Title: IFN-Dependent and -Independent Reduction in West Nile Virus Infectivity in Human Dermal Fibroblasts

    Journal: Viruses

    doi: 10.3390/v6031424

    Antiviral response to WNV-AUS60 and WNV-NY in HDFs. ( A ) Steady state protein levels of WNV, ISG56, ISG15, Phospho-STAT-2, STAT-2, Phospho-STAT-1, STAT-1, IRF-9, and GAPDH in mock and WNV-infected (MOI = 0.05) HDF cells. Extracts prepared at the indicated times post-infection were examined by immunoblot. A representative example from three independent experiments is shown; ( B ) Sensitivity of HDFs to IFN. HDF cells were treated with 0, 0.625, 1.25, or 2.5 IU/mL IFN-β for 24 h prior to infection with vesicular stomatitis virus (VSV) (MOI = 1). Supernatants were collected at 24 h post-infection and VSV titers were determined by plaque assay on Vero cells. Values represent the average number of plaque forming units (PFU) per mL (±standard error) from at least three independent experiments. Statistical significance was determined by an unpaired t-test. Asterisks indicate differences that are statistically significant (*** p
    Figure Legend Snippet: Antiviral response to WNV-AUS60 and WNV-NY in HDFs. ( A ) Steady state protein levels of WNV, ISG56, ISG15, Phospho-STAT-2, STAT-2, Phospho-STAT-1, STAT-1, IRF-9, and GAPDH in mock and WNV-infected (MOI = 0.05) HDF cells. Extracts prepared at the indicated times post-infection were examined by immunoblot. A representative example from three independent experiments is shown; ( B ) Sensitivity of HDFs to IFN. HDF cells were treated with 0, 0.625, 1.25, or 2.5 IU/mL IFN-β for 24 h prior to infection with vesicular stomatitis virus (VSV) (MOI = 1). Supernatants were collected at 24 h post-infection and VSV titers were determined by plaque assay on Vero cells. Values represent the average number of plaque forming units (PFU) per mL (±standard error) from at least three independent experiments. Statistical significance was determined by an unpaired t-test. Asterisks indicate differences that are statistically significant (*** p

    Techniques Used: Infection, Plaque Assay

    17) Product Images from "Intestinal Myofibroblasts Produce Nitric Oxide in Response to Combinatorial Cytokine Stimulation"

    Article Title: Intestinal Myofibroblasts Produce Nitric Oxide in Response to Combinatorial Cytokine Stimulation

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.24164

    Combinatorial cytokine stimulation of IMF induces iNOS expression and NO production through a signaling pathway that requires activation of Akt,STAT1, and NF-κB. A: Western blots with antibodies specific to phosphorylated Ser473 on Akt, total
    Figure Legend Snippet: Combinatorial cytokine stimulation of IMF induces iNOS expression and NO production through a signaling pathway that requires activation of Akt,STAT1, and NF-κB. A: Western blots with antibodies specific to phosphorylated Ser473 on Akt, total

    Techniques Used: Expressing, Activation Assay, Western Blot

    18) Product Images from "Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation 1"

    Article Title: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation 1

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

    doi: 10.4049/jimmunol.0900331

    Adenosine has no effect on IFN- γ -induced STAT1 tyrosine phosphorylation in mouse macrophages. A , Whole-cell lysates were prepared and subjected to Western blot analysis. A representative immunoblot of phospho-STAT1 at Y701 is shown. M, media;
    Figure Legend Snippet: Adenosine has no effect on IFN- γ -induced STAT1 tyrosine phosphorylation in mouse macrophages. A , Whole-cell lysates were prepared and subjected to Western blot analysis. A representative immunoblot of phospho-STAT1 at Y701 is shown. M, media;

    Techniques Used: Western Blot

    Adenosine inhibits phosphorylation of STAT1 at S727 but not at Y701 in IFN- γ -treated human macrophages. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. THP-1 macrophages were treated
    Figure Legend Snippet: Adenosine inhibits phosphorylation of STAT1 at S727 but not at Y701 in IFN- γ -treated human macrophages. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. THP-1 macrophages were treated

    Techniques Used: Derivative Assay, Western Blot

    A 3 receptor stimulation is necessary for adenosine-mediated suppression of STAT1-dependent genes in activated macrophages. RAW 264.7 cells were treated for 4 h with IFN- γ (IFNg), IFN- γ plus Cl-IB-MECA, or IFN- γ plus Cl-IB-MECA
    Figure Legend Snippet: A 3 receptor stimulation is necessary for adenosine-mediated suppression of STAT1-dependent genes in activated macrophages. RAW 264.7 cells were treated for 4 h with IFN- γ (IFNg), IFN- γ plus Cl-IB-MECA, or IFN- γ plus Cl-IB-MECA

    Techniques Used:

    Adenosine reduces IFN- γ -induced phosphorylation at the STAT1 S727 residue in mouse macrophages. A , Whole-cell lysates were prepared and subjected to Western blot analysis. A representative immunoblot of phospho-STAT1 at S727 is shown. M, medium;
    Figure Legend Snippet: Adenosine reduces IFN- γ -induced phosphorylation at the STAT1 S727 residue in mouse macrophages. A , Whole-cell lysates were prepared and subjected to Western blot analysis. A representative immunoblot of phospho-STAT1 at S727 is shown. M, medium;

    Techniques Used: Western Blot

    Adenosine signaling has no effect on IFN- γ -induced STAT1 tyrosine phosphorylation in THP-1 cells. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. Treatments included media, IFN-
    Figure Legend Snippet: Adenosine signaling has no effect on IFN- γ -induced STAT1 tyrosine phosphorylation in THP-1 cells. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. Treatments included media, IFN-

    Techniques Used: Derivative Assay, Western Blot

    Adenosine treatment does not alter STAT1 DNA binding. Nuclear extract (12.5 μ g) from each sample was used in a transcription factor assay to determine STAT1 DNA binding. The assay was performed in the absence or presence of competitor oligonucleotide
    Figure Legend Snippet: Adenosine treatment does not alter STAT1 DNA binding. Nuclear extract (12.5 μ g) from each sample was used in a transcription factor assay to determine STAT1 DNA binding. The assay was performed in the absence or presence of competitor oligonucleotide

    Techniques Used: Binding Assay, Transcription Factor Assay

    Treatment of IFN- γ -stimulated RAW 264.7 macrophages with adenosine decreases STAT1-responsive promoter activity. Cells were transfected with a STAT1 homodimer reporter vector (GAS-Luc) mixed with a Renilla luciferase control. Twenty-four hours
    Figure Legend Snippet: Treatment of IFN- γ -stimulated RAW 264.7 macrophages with adenosine decreases STAT1-responsive promoter activity. Cells were transfected with a STAT1 homodimer reporter vector (GAS-Luc) mixed with a Renilla luciferase control. Twenty-four hours

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

    Selective stimulation of the A 3 receptor reduces STAT1 transcriptional activity in IFN- γ -treated macrophages. RAW 264.7 cells were cotransfected with a STAT1 homodimer reporter vector (GAS-Luc) mixed with a Renilla luciferase control. Twenty-four
    Figure Legend Snippet: Selective stimulation of the A 3 receptor reduces STAT1 transcriptional activity in IFN- γ -treated macrophages. RAW 264.7 cells were cotransfected with a STAT1 homodimer reporter vector (GAS-Luc) mixed with a Renilla luciferase control. Twenty-four

    Techniques Used: Activity Assay, Plasmid Preparation, Luciferase

    Adenosine signaling through the A 3 receptor suppresses IFN- γ -induced STAT1 S727 phosphorylation in human macrophages. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. Treatments included
    Figure Legend Snippet: Adenosine signaling through the A 3 receptor suppresses IFN- γ -induced STAT1 S727 phosphorylation in human macrophages. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. Treatments included

    Techniques Used: Derivative Assay, Western Blot

    A 3 receptor signaling reduces IFN- γ -induced phosphorylation of STAT1 at S727 but not Y701. Whole-cell lysates from RAW 264.7 macrophages were prepared and subjected to Western blot analysis. Treatments include medium, IFN- γ (IFNg), IFN-
    Figure Legend Snippet: A 3 receptor signaling reduces IFN- γ -induced phosphorylation of STAT1 at S727 but not Y701. Whole-cell lysates from RAW 264.7 macrophages were prepared and subjected to Western blot analysis. Treatments include medium, IFN- γ (IFNg), IFN-

    Techniques Used: Western Blot

    19) Product Images from "Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells"

    Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2019.00059

    Effect of GRK2 siRNA transfection on total and phosphorylation levels of STAT1 and STAT3 in LPS-stimulated MG6 cells. (A) Typical Western blots of phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT3 before and 6 h after 100 ng/ml LPS application in the presence of GRK2 siRNAs (siGRK2) or the negative control siRNAs (siCTL). Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. (B) Phosphorylated levels of STAT1 and STAT3 6 h after 100 ng/ml LPS application when siCTL or siGRK2 was transfected. (C) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then phospho-STAT1 and phospho-STAT3 6 h after 100 ng/ml LPS were detected by Western blot analysis. GAPDH and lamin B served as a cytoplasmic and a nuclear marker, respectively. Shown are representative Western blots from three independent experiments in which the same results were obtained. (D) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 100 ng/ml LPS application. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for three independent experiments. ∗ P
    Figure Legend Snippet: Effect of GRK2 siRNA transfection on total and phosphorylation levels of STAT1 and STAT3 in LPS-stimulated MG6 cells. (A) Typical Western blots of phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT3 before and 6 h after 100 ng/ml LPS application in the presence of GRK2 siRNAs (siGRK2) or the negative control siRNAs (siCTL). Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. (B) Phosphorylated levels of STAT1 and STAT3 6 h after 100 ng/ml LPS application when siCTL or siGRK2 was transfected. (C) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then phospho-STAT1 and phospho-STAT3 6 h after 100 ng/ml LPS were detected by Western blot analysis. GAPDH and lamin B served as a cytoplasmic and a nuclear marker, respectively. Shown are representative Western blots from three independent experiments in which the same results were obtained. (D) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 100 ng/ml LPS application. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for three independent experiments. ∗ P

    Techniques Used: Transfection, Western Blot, Negative Control, Expressing, Isolation, Marker

    Effect of GRK2 siRNA transfection on TLR3-mediated signaling for iNOS expression in MG6 cells. (A) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then the time course of changes in IRF1 levels in each fraction after 50 μg/ml poly(I:C) application was tracked by Western blot analysis. (B) Effect of transfection of GRK2 siRNAs (siGRK2) on nuclear translocation of IRF1 60 min after challenge with 50 μg/ml poly(I:C) was compared with that when the negative control siRNAs (siCTL) was transfected. GAPDH and lamin B served as a cytoplasmic and nuclear marker, respectively. (C) Effect of siGRK2 transfection on IFN-β mRNA expression levels 3 h after 50 μg/ml poly(I:C) application. (D) Effect of siGRK2 transfection on phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT-3 4 h after 50 μg/ml poly(I:C) application. GAPDH served as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (E) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 50 μg/ml poly(I:C) application. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for three independent experiments. ∗ P
    Figure Legend Snippet: Effect of GRK2 siRNA transfection on TLR3-mediated signaling for iNOS expression in MG6 cells. (A) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then the time course of changes in IRF1 levels in each fraction after 50 μg/ml poly(I:C) application was tracked by Western blot analysis. (B) Effect of transfection of GRK2 siRNAs (siGRK2) on nuclear translocation of IRF1 60 min after challenge with 50 μg/ml poly(I:C) was compared with that when the negative control siRNAs (siCTL) was transfected. GAPDH and lamin B served as a cytoplasmic and nuclear marker, respectively. (C) Effect of siGRK2 transfection on IFN-β mRNA expression levels 3 h after 50 μg/ml poly(I:C) application. (D) Effect of siGRK2 transfection on phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT-3 4 h after 50 μg/ml poly(I:C) application. GAPDH served as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (E) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 50 μg/ml poly(I:C) application. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for three independent experiments. ∗ P

    Techniques Used: Transfection, Expressing, Isolation, Western Blot, Translocation Assay, Negative Control, Marker

    Effect of GRK2 siRNA transfection on phosphorylation levels of STAT1 and STAT3 and mRNA levels of iNOS in MG6 cells supplemented with exogenous IFN-β. (A) Time course of changes in phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT3 after supplementation with 10 ng/ml IFN-β. Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (B) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 10 ng/ml IFN-β supplementation in the presence of 10 ng/ml LPS. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for four independent experiments. N.D., not detected. ∗ P
    Figure Legend Snippet: Effect of GRK2 siRNA transfection on phosphorylation levels of STAT1 and STAT3 and mRNA levels of iNOS in MG6 cells supplemented with exogenous IFN-β. (A) Time course of changes in phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT3 after supplementation with 10 ng/ml IFN-β. Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (B) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 10 ng/ml IFN-β supplementation in the presence of 10 ng/ml LPS. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for four independent experiments. N.D., not detected. ∗ P

    Techniques Used: Transfection, Expressing, Western Blot

    Effects of STAT1 siRNA transfection and stattic on protein expression levels of iNOS and total and phosphorylation levels of STAT1 in LPS-stimulated MG6 cells. (A) iNOS, phospho-STAT1 at Tyr-701, and total STAT1 before and 15 h after 100 ng/ml LPS application in the presence of or STAT1 siRNAs (siSTAT1) or the negative control siRNAs (siCTL). (B) Concentration-dependent effect of stattic (0.5–2 μM) on iNOS expression before and 15 h after 100 ng/ml LPS application. (C) Effects of siSTAT1 alone, stattic (2 μM) alone, and combination of siSTAT1 and stattic (1 μM) on iNOS, phosphorylated STAT1, and total STAT1 before and 15 h after 100 ng/ml LPS application. (D) Effect of STAT3 siRNAs (siSTAT3) on iNOS and STAT3 expression before and 15 h after 100 ng/ml LPS application. GAPDH served as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained.
    Figure Legend Snippet: Effects of STAT1 siRNA transfection and stattic on protein expression levels of iNOS and total and phosphorylation levels of STAT1 in LPS-stimulated MG6 cells. (A) iNOS, phospho-STAT1 at Tyr-701, and total STAT1 before and 15 h after 100 ng/ml LPS application in the presence of or STAT1 siRNAs (siSTAT1) or the negative control siRNAs (siCTL). (B) Concentration-dependent effect of stattic (0.5–2 μM) on iNOS expression before and 15 h after 100 ng/ml LPS application. (C) Effects of siSTAT1 alone, stattic (2 μM) alone, and combination of siSTAT1 and stattic (1 μM) on iNOS, phosphorylated STAT1, and total STAT1 before and 15 h after 100 ng/ml LPS application. (D) Effect of STAT3 siRNAs (siSTAT3) on iNOS and STAT3 expression before and 15 h after 100 ng/ml LPS application. GAPDH served as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained.

    Techniques Used: Transfection, Expressing, Negative Control, Concentration Assay, Western Blot

    Changes in protein expression levels of iNOS and total and phosphorylation levels of STAT1 and STAT3 in LPS-stimulated MG6 cells. (A) Typical Western blots of iNOS, phospho-STAT1 at Tyr-701, total STAT-1, phospho-STAT3 at Tyr-705, and total STAT-3 after challenge with 100 ng/ml LPS. GAPDH served as loading control. (B) Time course of changes in iNOS protein expression after LPS application. (C) Time course of changes in STAT1 phosphorylation after LPS application. (D) Time course of changes in STAT3 phosphorylation after LPS application. The results represent the mean ± SEM for three independent experiments. ∗ P
    Figure Legend Snippet: Changes in protein expression levels of iNOS and total and phosphorylation levels of STAT1 and STAT3 in LPS-stimulated MG6 cells. (A) Typical Western blots of iNOS, phospho-STAT1 at Tyr-701, total STAT-1, phospho-STAT3 at Tyr-705, and total STAT-3 after challenge with 100 ng/ml LPS. GAPDH served as loading control. (B) Time course of changes in iNOS protein expression after LPS application. (C) Time course of changes in STAT1 phosphorylation after LPS application. (D) Time course of changes in STAT3 phosphorylation after LPS application. The results represent the mean ± SEM for three independent experiments. ∗ P

    Techniques Used: Expressing, Western Blot

    Effect of GRK2 siRNA transfection on paclitaxel-induced TLR4 signaling for iNOS expression in MG6 cells. (A) Phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT-3 before and 6 h after 10 μM paclitaxel (PTX) application in the presence of GRK2 siRNAs (siGRK2) or the negative control siRNAs (siCTL). Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (B) Effect of siGRK2 transfection on iNOS protein expression 12 h after 5 μM paclitaxel application. The results represent the mean ± SEM for three independent experiments. ∗∗ P
    Figure Legend Snippet: Effect of GRK2 siRNA transfection on paclitaxel-induced TLR4 signaling for iNOS expression in MG6 cells. (A) Phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT-3 before and 6 h after 10 μM paclitaxel (PTX) application in the presence of GRK2 siRNAs (siGRK2) or the negative control siRNAs (siCTL). Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (B) Effect of siGRK2 transfection on iNOS protein expression 12 h after 5 μM paclitaxel application. The results represent the mean ± SEM for three independent experiments. ∗∗ P

    Techniques Used: Transfection, Expressing, Negative Control, Western Blot

    20) Product Images from "Critical Role of IRF-3 in the Direct Regulation of dsRNA-Induced Retinoic Acid-Inducible Gene-I (RIG-I) Expression"

    Article Title: Critical Role of IRF-3 in the Direct Regulation of dsRNA-Induced Retinoic Acid-Inducible Gene-I (RIG-I) Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163520

    Promoter analysis of the human RIG-I gene. (A) Putative consensus sequences of STAT1, ISRE, c-Rel and IRF-E on the RIG-I promoter are shown. (B) and (C) HeLa cells were co-transfected with pGL4.11 (empty) or serial human RIG-I luciferase reporter constructs and Renilla luciferase expression vector (pGL4.74) for 24 h. The cells were further transfected with poly I:C (100 ng) for 4 h. The reporter activities are shown as relative values, specifically ratios of the firefly luciferase activities driven by the RIG-I promoters to the Renilla luciferase activities. The means (±SD) of three experiments are shown; † P
    Figure Legend Snippet: Promoter analysis of the human RIG-I gene. (A) Putative consensus sequences of STAT1, ISRE, c-Rel and IRF-E on the RIG-I promoter are shown. (B) and (C) HeLa cells were co-transfected with pGL4.11 (empty) or serial human RIG-I luciferase reporter constructs and Renilla luciferase expression vector (pGL4.74) for 24 h. The cells were further transfected with poly I:C (100 ng) for 4 h. The reporter activities are shown as relative values, specifically ratios of the firefly luciferase activities driven by the RIG-I promoters to the Renilla luciferase activities. The means (±SD) of three experiments are shown; † P

    Techniques Used: Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

    STAT1- and type I IFN-independent RIG-I expression in response to dsRNA. 2fTGH, U3A, and U5A cells were transfected with poly I:C (100 ng) and incubated for up to 8 h. The expression levels of RIG-I were determined by quantitative RT-PCR. The means (±SD) of three experiments are shown.
    Figure Legend Snippet: STAT1- and type I IFN-independent RIG-I expression in response to dsRNA. 2fTGH, U3A, and U5A cells were transfected with poly I:C (100 ng) and incubated for up to 8 h. The expression levels of RIG-I were determined by quantitative RT-PCR. The means (±SD) of three experiments are shown.

    Techniques Used: Expressing, Transfection, Incubation, Quantitative RT-PCR

    Proposal model for the direct role of IRF-3 in both constitutive and induced RIG-I expression. Upon viral infection, a low level of constitutively expressed RIG-I recognizes viral RNA, inducing the cells to reach an antiviral state. In the antiviral state, IRF-3 is phosphorylated in response to RLR signaling and translocates to the nucleus to induce type I IFNs. Our findings indicate that activated IRF-3 is also able to directly enhance the expression of RIG-I to enhance antiviral signaling. Infected cells produce IFNs, which subsequently activate STAT1, leading to the robust expression of RIG-I in neighboring cells.
    Figure Legend Snippet: Proposal model for the direct role of IRF-3 in both constitutive and induced RIG-I expression. Upon viral infection, a low level of constitutively expressed RIG-I recognizes viral RNA, inducing the cells to reach an antiviral state. In the antiviral state, IRF-3 is phosphorylated in response to RLR signaling and translocates to the nucleus to induce type I IFNs. Our findings indicate that activated IRF-3 is also able to directly enhance the expression of RIG-I to enhance antiviral signaling. Infected cells produce IFNs, which subsequently activate STAT1, leading to the robust expression of RIG-I in neighboring cells.

    Techniques Used: Expressing, Infection

    STAT1- and type I IFN-independent RIG-I expression requires IRF3. HeLa (A), and 2fTGH, U3A, and U5A cells (B) were transfected with siRNA against IRF-1 or IRF-3 or control siRNA for 48 h and then transfected with poly I:C (100 ng) for 4 h. The expression levels of RIG-I were determined by quantitative RT-PCR. The means (±SD) of three experiments are shown; † P
    Figure Legend Snippet: STAT1- and type I IFN-independent RIG-I expression requires IRF3. HeLa (A), and 2fTGH, U3A, and U5A cells (B) were transfected with siRNA against IRF-1 or IRF-3 or control siRNA for 48 h and then transfected with poly I:C (100 ng) for 4 h. The expression levels of RIG-I were determined by quantitative RT-PCR. The means (±SD) of three experiments are shown; † P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    21) Product Images from "Dexmedetomidine protects against renal ischemia and reperfusion injury by inhibiting the JAK/STAT signaling activation"

    Article Title: Dexmedetomidine protects against renal ischemia and reperfusion injury by inhibiting the JAK/STAT signaling activation

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-11-141

    Dexmedetomidine inhibited the phosphorylations of JAK2, STAT1 and STAT3. Representative Western blots for the phosphorylations of JAK2, STAT1, STAT3 and total JAK2, STAT1, STAT3 expression (A) of the kidneys were detected after 48 h of renal I/R in the sham, IRI, DMSO, Atip, DEX and AG490 groups. Densitometry analysis of Western blots for the ratio of p-JAK2/JAK2 (B) , p-STAT1/STAT1 (C) and p-STAT3/STAT3 (D) . Data were represented as mean ± SEM ( n = 8). * P
    Figure Legend Snippet: Dexmedetomidine inhibited the phosphorylations of JAK2, STAT1 and STAT3. Representative Western blots for the phosphorylations of JAK2, STAT1, STAT3 and total JAK2, STAT1, STAT3 expression (A) of the kidneys were detected after 48 h of renal I/R in the sham, IRI, DMSO, Atip, DEX and AG490 groups. Densitometry analysis of Western blots for the ratio of p-JAK2/JAK2 (B) , p-STAT1/STAT1 (C) and p-STAT3/STAT3 (D) . Data were represented as mean ± SEM ( n = 8). * P

    Techniques Used: Western Blot, Expressing

    The effect of dexmedetomidine on the expression of p-STAT1 in the kidneys following renal I/R-induced injury. Immunohistochemical staining of p-STAT1 was performed on formalin-fixed paraffin-embedded kidneys of the sham (A) , IRI (B) , DMSO (C) , Atip (D) , DEX (E) and AG490 (F) groups at 48 h following renal I/R. Bar = 50 μm. n = 8 per group.
    Figure Legend Snippet: The effect of dexmedetomidine on the expression of p-STAT1 in the kidneys following renal I/R-induced injury. Immunohistochemical staining of p-STAT1 was performed on formalin-fixed paraffin-embedded kidneys of the sham (A) , IRI (B) , DMSO (C) , Atip (D) , DEX (E) and AG490 (F) groups at 48 h following renal I/R. Bar = 50 μm. n = 8 per group.

    Techniques Used: Expressing, Immunohistochemistry, Staining, Formalin-fixed Paraffin-Embedded

    22) Product Images from "Structural Alterations of the FAS Gene in Cutaneous T-Cell Lymphoma (CTCL)"

    Article Title: Structural Alterations of the FAS Gene in Cutaneous T-Cell Lymphoma (CTCL)

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2010.10.020

    EMSA and supershift EMSA show reduced CTCL nuclear protein/STAT-1 binding to FAS promoter oligonucleotide probe bearing the -671 G SNP
    Figure Legend Snippet: EMSA and supershift EMSA show reduced CTCL nuclear protein/STAT-1 binding to FAS promoter oligonucleotide probe bearing the -671 G SNP

    Techniques Used: Binding Assay

    23) Product Images from "Gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1): Chronic mucocutaneous candidiasis accompanied by enamel defects and delayed dental shedding"

    Article Title: Gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1): Chronic mucocutaneous candidiasis accompanied by enamel defects and delayed dental shedding

    Journal: The Journal of Allergy and Clinical Immunology

    doi: 10.1016/j.jaci.2014.05.044

    The mutant T385M STAT1 allele is a gain-of-phosphorylation and gain-of-function mutation. A, Direct sequence analysis of exon 14 of STAT1 (forward sequence) in a control subject and the patient with a c.1153C > T resulting in p.T385M. B, Intracellular staining of phosphorylated tyrosine 701 STAT1 (STAT1p) in lymphocytes after stimulation with IFN-γ (2000 IU/mL, left panel ) or IFN-α (10 5 IU/mL, right panel ) for 15 minutes. STAT1 and STATp are shown in a control subject (red) and in the T385M patient (blue) . Unstimulated conditions are represented as dashed lines . Results shown are representative of 2 independent experiments. MFI , Mean fluorescence intensity. C, Evaluation of STAT1, STAT1 phosphorylation, and STAT1p GAS DNA-binding capacity. Fibroblasts derived from wild-type (WT)/WT control subjects (C1 and C2), p.T385M/WT (patient P1), and p.K388E/WT (patient P2) were stimulated with 100 U/mL IFN-α (α) or 100 U/mL IFN-γ (Υ) or left unstimulated (−) for 60 minutes. a , Western blotting was carried out for detection of STAT1 and STAT1p levels in nuclear extracts (5 μg per sample). Heterogeneous nuclear ribonucleoprotein I (hnRNP I) was used as a loading control reference. b , STAT1 GAS DNA-binding capacity was evaluated by using EMSA. One microgram of nuclear extract was preincubated with 20,000 cpm of GAS probe at room temperature before nondenaturing PAGE separating free from STAT-bound probe.
    Figure Legend Snippet: The mutant T385M STAT1 allele is a gain-of-phosphorylation and gain-of-function mutation. A, Direct sequence analysis of exon 14 of STAT1 (forward sequence) in a control subject and the patient with a c.1153C > T resulting in p.T385M. B, Intracellular staining of phosphorylated tyrosine 701 STAT1 (STAT1p) in lymphocytes after stimulation with IFN-γ (2000 IU/mL, left panel ) or IFN-α (10 5 IU/mL, right panel ) for 15 minutes. STAT1 and STATp are shown in a control subject (red) and in the T385M patient (blue) . Unstimulated conditions are represented as dashed lines . Results shown are representative of 2 independent experiments. MFI , Mean fluorescence intensity. C, Evaluation of STAT1, STAT1 phosphorylation, and STAT1p GAS DNA-binding capacity. Fibroblasts derived from wild-type (WT)/WT control subjects (C1 and C2), p.T385M/WT (patient P1), and p.K388E/WT (patient P2) were stimulated with 100 U/mL IFN-α (α) or 100 U/mL IFN-γ (Υ) or left unstimulated (−) for 60 minutes. a , Western blotting was carried out for detection of STAT1 and STAT1p levels in nuclear extracts (5 μg per sample). Heterogeneous nuclear ribonucleoprotein I (hnRNP I) was used as a loading control reference. b , STAT1 GAS DNA-binding capacity was evaluated by using EMSA. One microgram of nuclear extract was preincubated with 20,000 cpm of GAS probe at room temperature before nondenaturing PAGE separating free from STAT-bound probe.

    Techniques Used: Mutagenesis, Sequencing, Staining, Fluorescence, Binding Assay, Derivative Assay, Western Blot, Polyacrylamide Gel Electrophoresis

    24) Product Images from "Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells and mediates estrogen-induced apoptosis"

    Article Title: Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells and mediates estrogen-induced apoptosis

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-0916

    E 2 treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) 3 million MCF-7 and MCF-7:5C cells were bilaterally injected into the 4 th mammary fat pad of ovariectomized female NSG mice with and without E 2 capsule implants respectively. 35 days post-injection, mice were randomized into two groups with half of the E 2 capsule implants removed from MCF-7 injected mice and half of the MCF-7:5C injected mice received estrogen capsule implants. Tumors were measured using digital calipers and measurements were used to calculate tumor volume (mm 3 ) over time. ( b ) At the end of the experiment tumors were excised and final tumor volume was measured. In the MCF-7 injected mice, 12 tumors were collected from the −E 2 group and 11 tumors were collected from the +E 2 group. In the MCF-7:5C injected mice, 14 tumors were collected from the −E 2 group and 16 tumors were collected from the +E 2 group. ( c ) Cell death was analyzed by TUNEL staining and quantified by Image J software. ( d ) Hematoxylin and Eosin (H+E), IgG (negative staining control) and MUC1, P-STAT1, and IFITM1 staining in NSG mice from untreated and E 2 treated mice. *p
    Figure Legend Snippet: E 2 treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) 3 million MCF-7 and MCF-7:5C cells were bilaterally injected into the 4 th mammary fat pad of ovariectomized female NSG mice with and without E 2 capsule implants respectively. 35 days post-injection, mice were randomized into two groups with half of the E 2 capsule implants removed from MCF-7 injected mice and half of the MCF-7:5C injected mice received estrogen capsule implants. Tumors were measured using digital calipers and measurements were used to calculate tumor volume (mm 3 ) over time. ( b ) At the end of the experiment tumors were excised and final tumor volume was measured. In the MCF-7 injected mice, 12 tumors were collected from the −E 2 group and 11 tumors were collected from the +E 2 group. In the MCF-7:5C injected mice, 14 tumors were collected from the −E 2 group and 16 tumors were collected from the +E 2 group. ( c ) Cell death was analyzed by TUNEL staining and quantified by Image J software. ( d ) Hematoxylin and Eosin (H+E), IgG (negative staining control) and MUC1, P-STAT1, and IFITM1 staining in NSG mice from untreated and E 2 treated mice. *p

    Techniques Used: In Vivo, Injection, Mouse Assay, TUNEL Assay, Staining, Software, Negative Staining

    Rux treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) Whole cell lysates from MCF-7 and MCF-7:5C cells treated with 10 μmol Rux for 24 hours were immunoblotted for P-STAT1, STAT1 and IFITM1 protein expression. ( b ) 3 million MCF-7 or MCF-7:5C cells were injected into the 4 th mammary fat pad of female NSG mice. After 32 days of tumor growth, mice were randomized to treatment groups and half were given 50 mg/kg Rux by oral gavage every other day. At the end of the experiment tumors were excised and tumor volumes were determined by digital caliper measurement. In the MCF-7 injected mice, 8 tumors were collected from the -Rux group and 7 tumors were collected from the +Rux group. In the MCF-7:5C injected mice, 5 tumors were collected from the -Rux group and 6 tumors were collected from the +Rux group. ( c ) Tumors were subjected to TUNEL staining to quantify apoptotic cells. The intensity of TUNEL (red) staining was quantified using ten separate images with Image J Software ( bottom panel ). ( d ) Tumor expression of P-STAT1, STAT1 and IFITM1 after 21 days of treatment was determined by immunoblot. ( e ) Tumors were fixed, embedded in paraffin and sectioned onto glass slides. H+E staining revealed tumor architecture and MUC1, IFITM1, and P-STAT1 expression was determined by immunohistochemistry. *p
    Figure Legend Snippet: Rux treatment reduces tumor size and reduces MUC1, P-STAT1, and IFITM1 levels in AI-resistant cells in vivo . ( a ) Whole cell lysates from MCF-7 and MCF-7:5C cells treated with 10 μmol Rux for 24 hours were immunoblotted for P-STAT1, STAT1 and IFITM1 protein expression. ( b ) 3 million MCF-7 or MCF-7:5C cells were injected into the 4 th mammary fat pad of female NSG mice. After 32 days of tumor growth, mice were randomized to treatment groups and half were given 50 mg/kg Rux by oral gavage every other day. At the end of the experiment tumors were excised and tumor volumes were determined by digital caliper measurement. In the MCF-7 injected mice, 8 tumors were collected from the -Rux group and 7 tumors were collected from the +Rux group. In the MCF-7:5C injected mice, 5 tumors were collected from the -Rux group and 6 tumors were collected from the +Rux group. ( c ) Tumors were subjected to TUNEL staining to quantify apoptotic cells. The intensity of TUNEL (red) staining was quantified using ten separate images with Image J Software ( bottom panel ). ( d ) Tumor expression of P-STAT1, STAT1 and IFITM1 after 21 days of treatment was determined by immunoblot. ( e ) Tumors were fixed, embedded in paraffin and sectioned onto glass slides. H+E staining revealed tumor architecture and MUC1, IFITM1, and P-STAT1 expression was determined by immunohistochemistry. *p

    Techniques Used: In Vivo, Expressing, Injection, Mouse Assay, TUNEL Assay, Staining, Software, Immunohistochemistry

    MUC1 stabilizes JAK/STAT signaling which is necessary for IFITM1 expression. ( a ) T-47D, MCF-7, and MCF-7:5C cells were transiently transfected with siCon or siMUC1 for 72 hours and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( b ) T-47D, MCF-7, and MCF-7:5C cells were treated for 48 hours with Ruxolitinib (Rux) and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( c ) T-47D, MCF-7, and MCF-7:5C cells were treated for 24 hours with E 2 and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( d ) T-47D and MCF-7:5C cells were transiently transfected with control or MUC1 siRNA or treated with Rux. After 24 hours, mRNA expression of IFITM1 was determined by RT-PCR. ( e ) Whole cell lysates from T-47D and MCF-7:5C cells were treated with vehicle (Con) or 10 μmol Rux for 8 hours were immunoprecipitated with anti-MUC1 antibody or rabbit IgG and samples immunoblotted for MUC1, P-STAT2, P-STAT1 and ERα protein interaction. ( f ) Fixed T-47D and MCF-7:5C cell lysates were subjected to chromatin immunoprecipitation (ChIP) with antibodies against STAT1, STAT2, MUC1 or species-specific IgG control. As indicated, fixed whole cell lysates from T-47D and MCF-7:5C cells treated with 10 μmol Rux for 24 hours. qPCR was performed on the isolated DNA using primers designed to amplify the ISRE regulatory regions. Recruitment of the indicated proteins to the ISRE site was compared to input and IgG DNA and displayed as mean ± SD of technical triplicates in two independent experiments. Fold change between control and treatment is displayed above the indicated protein. ***p
    Figure Legend Snippet: MUC1 stabilizes JAK/STAT signaling which is necessary for IFITM1 expression. ( a ) T-47D, MCF-7, and MCF-7:5C cells were transiently transfected with siCon or siMUC1 for 72 hours and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( b ) T-47D, MCF-7, and MCF-7:5C cells were treated for 48 hours with Ruxolitinib (Rux) and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( c ) T-47D, MCF-7, and MCF-7:5C cells were treated for 24 hours with E 2 and immunoblotted for MUC1, P-STAT2, STAT2, P-STAT1, STAT1, and IFITM1 expression. ( d ) T-47D and MCF-7:5C cells were transiently transfected with control or MUC1 siRNA or treated with Rux. After 24 hours, mRNA expression of IFITM1 was determined by RT-PCR. ( e ) Whole cell lysates from T-47D and MCF-7:5C cells were treated with vehicle (Con) or 10 μmol Rux for 8 hours were immunoprecipitated with anti-MUC1 antibody or rabbit IgG and samples immunoblotted for MUC1, P-STAT2, P-STAT1 and ERα protein interaction. ( f ) Fixed T-47D and MCF-7:5C cell lysates were subjected to chromatin immunoprecipitation (ChIP) with antibodies against STAT1, STAT2, MUC1 or species-specific IgG control. As indicated, fixed whole cell lysates from T-47D and MCF-7:5C cells treated with 10 μmol Rux for 24 hours. qPCR was performed on the isolated DNA using primers designed to amplify the ISRE regulatory regions. Recruitment of the indicated proteins to the ISRE site was compared to input and IgG DNA and displayed as mean ± SD of technical triplicates in two independent experiments. Fold change between control and treatment is displayed above the indicated protein. ***p

    Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation

    25) Product Images from "Osteopontin Mediates Stat1 Degradation To Inhibit iNOS Transcription In A Cecal Ligation and Puncture Model of Sepsis"

    Article Title: Osteopontin Mediates Stat1 Degradation To Inhibit iNOS Transcription In A Cecal Ligation and Puncture Model of Sepsis

    Journal: Surgery

    doi: 10.1016/j.surg.2008.03.007

    Co-Immunoprecipitation and ubiquitination of P-STAT1 in CLP Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Blot is representative of four experiments.
    Figure Legend Snippet: Co-Immunoprecipitation and ubiquitination of P-STAT1 in CLP Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Blot is representative of four experiments.

    Techniques Used: Immunoprecipitation, Centrifugation, Incubation

    IP studies for Ub-Stat1 and iNOS in ex vivo treated BMM from OPN null and WT animals Ex vivo gain-of-function studies were performed using BMM from WT and OPN null mice. MG132 (10 µM), an inhibitor of 26S proteasome function, and/or exogenous OPN (50 µM) were added to assess Ub-P-STAT1 and iNOS expression. BMM cells were treated with LPS (50 ng/ml). The incubation period was 12 hours. Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Alternatively, IB was performed for iNOS. Blot is representative of four experiments.
    Figure Legend Snippet: IP studies for Ub-Stat1 and iNOS in ex vivo treated BMM from OPN null and WT animals Ex vivo gain-of-function studies were performed using BMM from WT and OPN null mice. MG132 (10 µM), an inhibitor of 26S proteasome function, and/or exogenous OPN (50 µM) were added to assess Ub-P-STAT1 and iNOS expression. BMM cells were treated with LPS (50 ng/ml). The incubation period was 12 hours. Cells were lysed and cleared by centrifugation. Aliquots were precleared by addition of normal IgG and 20 µl of appropriate agarose conjugate. After centrifugation, the supernatant was collected and incubated with 2 µg of P-Stat1 Ab. After washing, immunoblotting was performed using Ub AB. Alternatively, IB was performed for iNOS. Blot is representative of four experiments.

    Techniques Used: Ex Vivo, Mouse Assay, Expressing, Incubation, Centrifugation

    Western blot of iNOS, phosphorylated STAT1 (P-STAT1) and osteopontin (OPN) expression in CLP in WT and OPN null animals at 0h, 12h and 24h . Blot is presentative of four experiments.
    Figure Legend Snippet: Western blot of iNOS, phosphorylated STAT1 (P-STAT1) and osteopontin (OPN) expression in CLP in WT and OPN null animals at 0h, 12h and 24h . Blot is presentative of four experiments.

    Techniques Used: Western Blot, Expressing

    26) Product Images from "Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii"

    Article Title: Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2016.54.1.31

    Afatinib affects the phosphorylation of Stat6 activated by T. gondii (RH) in ARPE-19 cells. (A) The phosphorylation of the Tyr641 site of STAT6, but not the Tyr701 site of STAT1 in the ARPE-19 cells after challenge with T. gondii RH tachyzoites by western blot. The time of Afatinib challenge was the same as T. gondii (RH) infection. Afatinib challenge was at 1 hr before infection and at 1 hr post infection. The concentration of Afatinib was 10 μM, and DMSO was used as the vehicle. (B) Immunofluorescence stain at 2 hr and 8 hr post infection. The corresponding results of the western blot in Fig. 4A are marked in red frame.
    Figure Legend Snippet: Afatinib affects the phosphorylation of Stat6 activated by T. gondii (RH) in ARPE-19 cells. (A) The phosphorylation of the Tyr641 site of STAT6, but not the Tyr701 site of STAT1 in the ARPE-19 cells after challenge with T. gondii RH tachyzoites by western blot. The time of Afatinib challenge was the same as T. gondii (RH) infection. Afatinib challenge was at 1 hr before infection and at 1 hr post infection. The concentration of Afatinib was 10 μM, and DMSO was used as the vehicle. (B) Immunofluorescence stain at 2 hr and 8 hr post infection. The corresponding results of the western blot in Fig. 4A are marked in red frame.

    Techniques Used: Western Blot, Infection, Concentration Assay, Immunofluorescence, Staining

    27) Product Images from "Resveratrol Modulates Cytokine-Induced JAK/STAT Activation More Efficiently than 5-Aminosalicylic Acid: An In Vitro Approach"

    Article Title: Resveratrol Modulates Cytokine-Induced JAK/STAT Activation More Efficiently than 5-Aminosalicylic Acid: An In Vitro Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109048

    Resveratrol decreases activated-STAT1 levels in the nucleus of cytokine-stimulated HT-29 cells more efficiently than 5-ASA. Cells were pre-incubated with 25 µM Resv or 500 µM 5-ASA or both (25 µM Resv plus 500 µM 5-ASA) and then exposed to a combination of cytokines for 30 minutes. The levels of Tyr701 phospho-STAT1 were analyzed in nuclear extracts by Western blotting (A), as described in “ Materials and Methods ” and expressed as percentage of cytokine-stimulated cells. Values are mean ± SEM of at least three independent experiments, each one in duplicate. *** P
    Figure Legend Snippet: Resveratrol decreases activated-STAT1 levels in the nucleus of cytokine-stimulated HT-29 cells more efficiently than 5-ASA. Cells were pre-incubated with 25 µM Resv or 500 µM 5-ASA or both (25 µM Resv plus 500 µM 5-ASA) and then exposed to a combination of cytokines for 30 minutes. The levels of Tyr701 phospho-STAT1 were analyzed in nuclear extracts by Western blotting (A), as described in “ Materials and Methods ” and expressed as percentage of cytokine-stimulated cells. Values are mean ± SEM of at least three independent experiments, each one in duplicate. *** P

    Techniques Used: Incubation, Western Blot

    28) Product Images from "Impaired activation of Stat1 and c-Jun as a possible defect in macrophages of patients with active tuberculosis"

    Article Title: Impaired activation of Stat1 and c-Jun as a possible defect in macrophages of patients with active tuberculosis

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2009.03985.x

    Stat1-dependent interferon (IFN)-γ signalling is accomplished in Mycobacterium tuberculosis -infected macrophages of patients with active tuberculosis (TB) as healthy normal subjects. Ten micrograms of nuclear extracts were incubated with 32 P-labelled DNA probe containing the gamma interferon activating sequence (GAS) and then assayed for electrophoretic mobility shift assay (EMSA) (a). Stat1-GAS specific binding was corroborated by competing with the same unlabelled probe (cold probe) or with a probe mutated for GAS site (mutant probe) or by using an anti-Stat1 antibody to produce a supershift (SS) in EMSAs of nuclear extracts of macrophages incubated 45 min with IFN-γ (b). Stat1 phosphorylation (Tyr701) was corroborated by Western blotting of nuclear extracts (c). EMSAs were performed with 10 µg of nuclear extracts of M. tuberculosis -infected macrophages (48 h) incubated for 45 min with IFN-γ in patients with active TB (TBP, n = 10), healthy household contacts (HHC, n = 10) and healthy uninfected subjects (CC, n = 12) (d).
    Figure Legend Snippet: Stat1-dependent interferon (IFN)-γ signalling is accomplished in Mycobacterium tuberculosis -infected macrophages of patients with active tuberculosis (TB) as healthy normal subjects. Ten micrograms of nuclear extracts were incubated with 32 P-labelled DNA probe containing the gamma interferon activating sequence (GAS) and then assayed for electrophoretic mobility shift assay (EMSA) (a). Stat1-GAS specific binding was corroborated by competing with the same unlabelled probe (cold probe) or with a probe mutated for GAS site (mutant probe) or by using an anti-Stat1 antibody to produce a supershift (SS) in EMSAs of nuclear extracts of macrophages incubated 45 min with IFN-γ (b). Stat1 phosphorylation (Tyr701) was corroborated by Western blotting of nuclear extracts (c). EMSAs were performed with 10 µg of nuclear extracts of M. tuberculosis -infected macrophages (48 h) incubated for 45 min with IFN-γ in patients with active TB (TBP, n = 10), healthy household contacts (HHC, n = 10) and healthy uninfected subjects (CC, n = 12) (d).

    Techniques Used: Infection, Incubation, Sequencing, Electrophoretic Mobility Shift Assay, Binding Assay, Mutagenesis, Western Blot

    29) Product Images from "Curative Effects of Thiacremonone against Acetaminophen-Induced Acute Hepatic Failure via Inhibition of Proinflammatory Cytokines Production and Infiltration of Cytotoxic Immune Cells and Kupffer Cells"

    Article Title: Curative Effects of Thiacremonone against Acetaminophen-Induced Acute Hepatic Failure via Inhibition of Proinflammatory Cytokines Production and Infiltration of Cytotoxic Immune Cells and Kupffer Cells

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/974794

    Reduction of NF- κ B and STAT1 in the liver of thiacremonone pretreated mice. (a) The expression of p50 and p65 phosphorylation in nuclear extracts (NE) was determined by Western blotting, and the DNA binding activity of NF- κ B was determined in the nuclear extracts of APAP-treated mice and thiacremonone pretreated mice liver tissues by EMSA. (b) The expression of p-STAT1 and STAT1 in NE was determined by Western blotting and the DNA binding activity of STAT1 was determined in the nuclear extracts of APAP-treated mice and thiacremonone pretreated mice liver tissues by EMSA.
    Figure Legend Snippet: Reduction of NF- κ B and STAT1 in the liver of thiacremonone pretreated mice. (a) The expression of p50 and p65 phosphorylation in nuclear extracts (NE) was determined by Western blotting, and the DNA binding activity of NF- κ B was determined in the nuclear extracts of APAP-treated mice and thiacremonone pretreated mice liver tissues by EMSA. (b) The expression of p-STAT1 and STAT1 in NE was determined by Western blotting and the DNA binding activity of STAT1 was determined in the nuclear extracts of APAP-treated mice and thiacremonone pretreated mice liver tissues by EMSA.

    Techniques Used: Mouse Assay, Expressing, Western Blot, Binding Assay, Activity Assay

    30) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284190

    STAT1 co-immunoprecipitates with DOT1L. A–C , whole cell extracts prepared from 2fTGH ( A ), shRNAmir-DOT1L ( shRNA-DOT1L ) ( B ), or shRNAmir non-silencing ( shRNA-NS ) ( C ) cells were immunoprecipitated ( IP ) with α-DOT1L, α-STAT1, or IgG (negative control) and then immunoblotted ( IB ) with the indicated antibodies. A 5% input aliquot of the extracts was included for reference.
    Figure Legend Snippet: STAT1 co-immunoprecipitates with DOT1L. A–C , whole cell extracts prepared from 2fTGH ( A ), shRNAmir-DOT1L ( shRNA-DOT1L ) ( B ), or shRNAmir non-silencing ( shRNA-NS ) ( C ) cells were immunoprecipitated ( IP ) with α-DOT1L, α-STAT1, or IgG (negative control) and then immunoblotted ( IB ) with the indicated antibodies. A 5% input aliquot of the extracts was included for reference.

    Techniques Used: shRNA, Immunoprecipitation, Negative Control

    Mapping of the DOT1L region that interacts with STAT1 in GST pulldown assays. A , graphic depiction of the DOT1L fragments and their relative STAT1 binding affinities. The presence (+) or absence (−) of an interaction is shown, with +, ++, and +++ indicating weak, modest, and strong interactions, respectively. N-term , N-terminal; C-term , C-terminal. B , GST pulldown assays using whole extracts prepared from 2fTGH cells transiently transfected with pcDNA3β FLAG-tagged DOT1L or FLAG-tagged DOT1L fragment vectors and then immunoblotted ( IB ) with α-FLAG. A 5% input aliquot of the extracts was included for reference.
    Figure Legend Snippet: Mapping of the DOT1L region that interacts with STAT1 in GST pulldown assays. A , graphic depiction of the DOT1L fragments and their relative STAT1 binding affinities. The presence (+) or absence (−) of an interaction is shown, with +, ++, and +++ indicating weak, modest, and strong interactions, respectively. N-term , N-terminal; C-term , C-terminal. B , GST pulldown assays using whole extracts prepared from 2fTGH cells transiently transfected with pcDNA3β FLAG-tagged DOT1L or FLAG-tagged DOT1L fragment vectors and then immunoblotted ( IB ) with α-FLAG. A 5% input aliquot of the extracts was included for reference.

    Techniques Used: Binding Assay, Transfection

    RNAi-mediated depletion of DOT1L decreases IRF1 gene expression. A , DOT1L Western blot of whole cell extracts collected from 2fTGH cells stably expressing the pTRIPZ shRNAmir non-silencing ( shRNA-NS ) or shRNAmir-DOT1L ( shRNA-DOT1L ) vectors with or without IFN-γ. Dynamin served as a loading control. B and C , qRT-PCR to quantitate IRF1 mRNA and heteronuclear RNA ( hnRNA ) levels in the shRNAmir non-silencing and shRNAmir-DOT1L cell lines treated with IFN-γ for the times indicated or left untreated ( Un ). IRF1 was normalized to GAPDH , and expression is presented as -fold change relative to the uninduced, shRNAmir-NS condition. Error bars indicate S.E. ( n = 3). Student's t test determined significance; **, p ≤ 0.01, *, p ≤ 0.05. D , IRF1 Western blot of whole cell extracts of shRNAmir non-silencing or shRNAmir-DOT1L cells treated with IFN-γ for the indicated times or left untreated. GAPDH served as a loading control. E , H3K79me3 Western blot of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines. Pan H3 served as a loading control. F , STAT1 and phospho-STAT1 Western blots of whole cell extracts collected from 2fTGH cells stably expressing the shRNAmir non-silencing or shRNAmir-DOT1L vectors. The ratio of phospho-STAT1 ( pSTAT ) to total STAT1 is the same in both cell lines. Western blot bands were quantified with ImageJ.
    Figure Legend Snippet: RNAi-mediated depletion of DOT1L decreases IRF1 gene expression. A , DOT1L Western blot of whole cell extracts collected from 2fTGH cells stably expressing the pTRIPZ shRNAmir non-silencing ( shRNA-NS ) or shRNAmir-DOT1L ( shRNA-DOT1L ) vectors with or without IFN-γ. Dynamin served as a loading control. B and C , qRT-PCR to quantitate IRF1 mRNA and heteronuclear RNA ( hnRNA ) levels in the shRNAmir non-silencing and shRNAmir-DOT1L cell lines treated with IFN-γ for the times indicated or left untreated ( Un ). IRF1 was normalized to GAPDH , and expression is presented as -fold change relative to the uninduced, shRNAmir-NS condition. Error bars indicate S.E. ( n = 3). Student's t test determined significance; **, p ≤ 0.01, *, p ≤ 0.05. D , IRF1 Western blot of whole cell extracts of shRNAmir non-silencing or shRNAmir-DOT1L cells treated with IFN-γ for the indicated times or left untreated. GAPDH served as a loading control. E , H3K79me3 Western blot of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines. Pan H3 served as a loading control. F , STAT1 and phospho-STAT1 Western blots of whole cell extracts collected from 2fTGH cells stably expressing the shRNAmir non-silencing or shRNAmir-DOT1L vectors. The ratio of phospho-STAT1 ( pSTAT ) to total STAT1 is the same in both cell lines. Western blot bands were quantified with ImageJ.

    Techniques Used: Expressing, Western Blot, Stable Transfection, shRNA, Quantitative RT-PCR

    Overexpression of the SID of DOT1L represses IRF1 gene expression and alters STAT1 binding. A , qRT-PCR measured IRF1 mRNA expression in 2fTGH, shRNAmir non-silencing ( shRNA-NS ), and shRNAmir-DOT1L ( shRNA-DOT1L ) cell lines that were transiently transfected with pcDNA3.0 DOT1L SID, ΔC, ΔN, or empty vector. IFN-γ induction was for 30 min. IRF1 was normalized to GAPDH , and expression is presented as -fold change relative to the uninduced, shRNAmir-NS cell line transfected with empty vector. Error bars indicate S.E. ( n = 2). Student's t test determined significance, **, p ≤ 0.01, *, p ≤ 0.05. B , Western blots of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines that were transiently transfected as in panel A . PanH3 served as a loading control. C–H , ChIP, using the indicated antibodies in shRNAmir-DOT1L ( right panels ) or shRNAmir non-silencing ( left panels ) cells transiently transfected with pcDNA3.0 DOTL1 SID, ΔC, ΔN, or empty vector and treated with IFN-γ for 30 min. p ≤ 0.05 for black lines with an X or gray squares versus black lines with black squares or white squares in panel C ; black lines with gray squares versus others in panel D ; black lines with an X or white squares versus black lines with black squares or gray squares in panel E ; black lines with white squares versus others in panel F .
    Figure Legend Snippet: Overexpression of the SID of DOT1L represses IRF1 gene expression and alters STAT1 binding. A , qRT-PCR measured IRF1 mRNA expression in 2fTGH, shRNAmir non-silencing ( shRNA-NS ), and shRNAmir-DOT1L ( shRNA-DOT1L ) cell lines that were transiently transfected with pcDNA3.0 DOT1L SID, ΔC, ΔN, or empty vector. IFN-γ induction was for 30 min. IRF1 was normalized to GAPDH , and expression is presented as -fold change relative to the uninduced, shRNAmir-NS cell line transfected with empty vector. Error bars indicate S.E. ( n = 2). Student's t test determined significance, **, p ≤ 0.01, *, p ≤ 0.05. B , Western blots of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines that were transiently transfected as in panel A . PanH3 served as a loading control. C–H , ChIP, using the indicated antibodies in shRNAmir-DOT1L ( right panels ) or shRNAmir non-silencing ( left panels ) cells transiently transfected with pcDNA3.0 DOTL1 SID, ΔC, ΔN, or empty vector and treated with IFN-γ for 30 min. p ≤ 0.05 for black lines with an X or gray squares versus black lines with black squares or white squares in panel C ; black lines with gray squares versus others in panel D ; black lines with an X or white squares versus black lines with black squares or gray squares in panel E ; black lines with white squares versus others in panel F .

    Techniques Used: Over Expression, Expressing, Binding Assay, Quantitative RT-PCR, shRNA, Transfection, Plasmid Preparation, Western Blot, Chromatin Immunoprecipitation

    RNA polymerase II and STAT1 localization to the IRF1 promoter is reduced in shRNAmir-DOT1L cells. A–H , ChIP of shRNAmir non-silencing ( shRNA-NS ) or shRNAmir-DOT1L ( shRNA-DOT1L ) cells treated with IFN-γ for 30 min ( right panels ) or left untreated ( left panels ). ChIP was performed with the antibodies indicated, and qPCR, using primers spanning the IRF1 gene locus, quantified the precipitate yield reported as the percentage of input. IgG served as the negative control. p ≤ 0.05 for panels A, B , D , F , and H for solid lines with black diamonds versus dotted lines with black diamonds .
    Figure Legend Snippet: RNA polymerase II and STAT1 localization to the IRF1 promoter is reduced in shRNAmir-DOT1L cells. A–H , ChIP of shRNAmir non-silencing ( shRNA-NS ) or shRNAmir-DOT1L ( shRNA-DOT1L ) cells treated with IFN-γ for 30 min ( right panels ) or left untreated ( left panels ). ChIP was performed with the antibodies indicated, and qPCR, using primers spanning the IRF1 gene locus, quantified the precipitate yield reported as the percentage of input. IgG served as the negative control. p ≤ 0.05 for panels A, B , D , F , and H for solid lines with black diamonds versus dotted lines with black diamonds .

    Techniques Used: Chromatin Immunoprecipitation, shRNA, Real-time Polymerase Chain Reaction, Negative Control

    31) Product Images from "Double-Stranded RNA Induces Biphasic STAT1 Phosphorylation by both Type I Interferon (IFN)-Dependent and Type I IFN-Independent Pathways"

    Article Title: Double-Stranded RNA Induces Biphasic STAT1 Phosphorylation by both Type I Interferon (IFN)-Dependent and Type I IFN-Independent Pathways

    Journal: Journal of Virology

    doi: 10.1128/JVI.01881-12

    Influence of a neutralizing IFN receptor antibody on STAT1 phosphorylation in response to poly(I:C). (A) Following pretreatment with the anti-IFNAR neutralizing antibody (2.5 μg/well) for 1 h, A549 cells were transfected with poly(I:C) (200 ng)
    Figure Legend Snippet: Influence of a neutralizing IFN receptor antibody on STAT1 phosphorylation in response to poly(I:C). (A) Following pretreatment with the anti-IFNAR neutralizing antibody (2.5 μg/well) for 1 h, A549 cells were transfected with poly(I:C) (200 ng)

    Techniques Used: Transfection

    Effect of MAVS on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (MAVS) or a negative control, lacZ -silenced cells (Lac), were transfected with RIG-I-CARD (CARD) or an empty vector (mock) for the indicated periods.
    Figure Legend Snippet: Effect of MAVS on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (MAVS) or a negative control, lacZ -silenced cells (Lac), were transfected with RIG-I-CARD (CARD) or an empty vector (mock) for the indicated periods.

    Techniques Used: Stable Transfection, Negative Control, Transfection, Plasmid Preparation

    Mechanism of dsRNA-mediated STAT1 phosphorylation. (A) Our results indicate that the initial phosphorylation of STAT1 is RIG-I-MAVS dependent. MDA-5 is not involved in the initial dsRNA-induced STAT1 phosphorylation. dsRNA-induced type I IFN is essential
    Figure Legend Snippet: Mechanism of dsRNA-mediated STAT1 phosphorylation. (A) Our results indicate that the initial phosphorylation of STAT1 is RIG-I-MAVS dependent. MDA-5 is not involved in the initial dsRNA-induced STAT1 phosphorylation. dsRNA-induced type I IFN is essential

    Techniques Used: Multiple Displacement Amplification

    Involvement of RIG-I, MDA-5, and MAVS in STAT1 phosphorylation in response to poly(I:C). A549 cells were transfected with control siRNA or gene-specific siRNAs against RIG-I, MDA-5, or MAVS for 48 h. After the incubation, the cells were transfected with
    Figure Legend Snippet: Involvement of RIG-I, MDA-5, and MAVS in STAT1 phosphorylation in response to poly(I:C). A549 cells were transfected with control siRNA or gene-specific siRNAs against RIG-I, MDA-5, or MAVS for 48 h. After the incubation, the cells were transfected with

    Techniques Used: Multiple Displacement Amplification, Transfection, Incubation

    Influence of the IFN receptor on ISG induction. (A) U5A cells were transfected with poly(I:C) (200 ng) for 8 h or treated with IFN-γ (5 ng/ml) for 30 min, followed by fixation with 4% paraformaldehyde. STAT1 (green) and DAPI (blue; nuclei) stains
    Figure Legend Snippet: Influence of the IFN receptor on ISG induction. (A) U5A cells were transfected with poly(I:C) (200 ng) for 8 h or treated with IFN-γ (5 ng/ml) for 30 min, followed by fixation with 4% paraformaldehyde. STAT1 (green) and DAPI (blue; nuclei) stains

    Techniques Used: Transfection

    Kinetics of IFN-β production and STAT1 phosphorylation in response to poly(I:C) transfection. (A) A549 cells were transfected with poly(I:C) (200 ng), and the levels of IFN-β mRNA were determined by quantitative real-time PCR. Glyceraldehyde-3-phosphate
    Figure Legend Snippet: Kinetics of IFN-β production and STAT1 phosphorylation in response to poly(I:C) transfection. (A) A549 cells were transfected with poly(I:C) (200 ng), and the levels of IFN-β mRNA were determined by quantitative real-time PCR. Glyceraldehyde-3-phosphate

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction

    Effect of RIG-CARD on STAT1 phosphorylation in U5A cells. (A) The cells were transfected with either an empty control vector (mock) or a vector encoding the CARD of RIG-I for 24 h. The levels of pSTAT1, STAT1, and FLAG-RIG-CARD were analyzed by immunoblotting.
    Figure Legend Snippet: Effect of RIG-CARD on STAT1 phosphorylation in U5A cells. (A) The cells were transfected with either an empty control vector (mock) or a vector encoding the CARD of RIG-I for 24 h. The levels of pSTAT1, STAT1, and FLAG-RIG-CARD were analyzed by immunoblotting.

    Techniques Used: Transfection, Plasmid Preparation

    dsRNA can induce STAT1 phosphorylation in IFNAR-deficient cells. (A) Characterization of U5A cells. A549, 2fTGH, and U5A cells were treated with IFN-α2b (α2b; 200 pg/ml) or IFN-γ (γ; 5 ng/ml) for 30 min or left untreated
    Figure Legend Snippet: dsRNA can induce STAT1 phosphorylation in IFNAR-deficient cells. (A) Characterization of U5A cells. A549, 2fTGH, and U5A cells were treated with IFN-α2b (α2b; 200 pg/ml) or IFN-γ (γ; 5 ng/ml) for 30 min or left untreated

    Techniques Used:

    Time course of STAT1 phosphorylation following the transfection of A549 cells with vectors expressing RIG-I constructs. (A) A549 cells were transfected with FLC-RIG-I, RIG-I-CARD, or an empty control vector for up to 8 h. (B) A549 cells were transfected
    Figure Legend Snippet: Time course of STAT1 phosphorylation following the transfection of A549 cells with vectors expressing RIG-I constructs. (A) A549 cells were transfected with FLC-RIG-I, RIG-I-CARD, or an empty control vector for up to 8 h. (B) A549 cells were transfected

    Techniques Used: Transfection, Expressing, Construct, Plasmid Preparation

    Effect of an anti-IFNAR neutralizing antibody on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (M) or lacZ -silenced cells (L) were transfected with FLAG-RIG-I-CARD or an empty vector (mock) for the indicated periods
    Figure Legend Snippet: Effect of an anti-IFNAR neutralizing antibody on RIG-I-CARD-induced STAT1 phosphorylation. Either stably MAVS-silenced 293-flp cells (M) or lacZ -silenced cells (L) were transfected with FLAG-RIG-I-CARD or an empty vector (mock) for the indicated periods

    Techniques Used: Stable Transfection, Transfection, Plasmid Preparation

    32) Product Images from "IL-21 Contributes to JAK3/STAT3 Activation and Promotes Cell Growth in ALK-Positive Anaplastic Large Cell Lymphoma"

    Article Title: IL-21 Contributes to JAK3/STAT3 Activation and Promotes Cell Growth in ALK-Positive Anaplastic Large Cell Lymphoma

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2009.080982

    Effects of rIL-21 on JAK3, STAT3, and STAT1. A: Western blot studies revealed that treatment of Karpas 299 recombinant IL-21 protein (10 ng/ml) for 30 minutes increased the levels of pJAK3 and pSTAT3. No detectable change in pSTAT1 was noted. B: rIL-21-induced
    Figure Legend Snippet: Effects of rIL-21 on JAK3, STAT3, and STAT1. A: Western blot studies revealed that treatment of Karpas 299 recombinant IL-21 protein (10 ng/ml) for 30 minutes increased the levels of pJAK3 and pSTAT3. No detectable change in pSTAT1 was noted. B: rIL-21-induced

    Techniques Used: Western Blot, Recombinant

    Related Articles

    Chromatin Immunoprecipitation:

    Article Title: Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses
    Article Snippet: Download Figure S2, DOCX file, 0.6 MB STAT1 binding corresponds with differential ISG expression. (A) RNA expression of consensus ISGs linked to STAT1 following MERS-CoV infection. .. Values represent log2 FC relative to time-matched mocks. (B) Chromatin immunoprecipitation with antibodies against phospho-STAT1 (Santa Cruz) followed by qPCR of the 5′UTR of upregulated genes CXCL10 and IFIT1 or downregulated genes CFHR1 and APOL6 within the context of H5N1-VN1203 infection. ..

    Article Title: Type I Interferon induces binding of STAT1 to Bcl6: Divergent Roles of STAT-family transcription factors in the TFH cell genetic program
    Article Snippet: List of primers and probes from Applied Biosystems: mouse ACTB, 4352341E; Bcl6, Mm01342164_m1; Tbx21, Mm00450960_m1; Maf, Mm02581355-s1; Irf-4, Mm00516431-m1; Batf, Mm00479410-m1. .. Chromatin immunoprecipitation (ChIP)�sequencing (seq) experiments and data processing were performed as previously described ( , ) using anti-STAT1 antibody (Santa Cruz: sc-592). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses
    Article Snippet: Download Figure S2, DOCX file, 0.6 MB STAT1 binding corresponds with differential ISG expression. (A) RNA expression of consensus ISGs linked to STAT1 following MERS-CoV infection. .. Values represent log2 FC relative to time-matched mocks. (B) Chromatin immunoprecipitation with antibodies against phospho-STAT1 (Santa Cruz) followed by qPCR of the 5′UTR of upregulated genes CXCL10 and IFIT1 or downregulated genes CFHR1 and APOL6 within the context of H5N1-VN1203 infection. ..

    Infection:

    Article Title: Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses
    Article Snippet: Download Figure S2, DOCX file, 0.6 MB STAT1 binding corresponds with differential ISG expression. (A) RNA expression of consensus ISGs linked to STAT1 following MERS-CoV infection. .. Values represent log2 FC relative to time-matched mocks. (B) Chromatin immunoprecipitation with antibodies against phospho-STAT1 (Santa Cruz) followed by qPCR of the 5′UTR of upregulated genes CXCL10 and IFIT1 or downregulated genes CFHR1 and APOL6 within the context of H5N1-VN1203 infection. ..

    other:

    Article Title: PHOSPHORYLATION OF Y372 IS CRITICAL FOR JAK2 TYROSINE KINASE ACTIVATION
    Article Snippet: To demonstrate equal STAT1 precipitation across all samples, the membrane was stripped and re-blotted with anti-STAT1 antibody ( ).

    Incubation:

    Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation
    Article Snippet: .. A 1/10 volume of the solution was used as input, and a 1/3 volume of the solution was precleared with protein G/salmon sperm DNA at 4°C for 4 h before being incubated with 6 μg of anti-STAT1 antibody (E23; Santa Cruz) or control rabbit IgG (Abcam) at 4°C. .. After 20 h of incubation, 25 μl of protein G/salmon sperm DNA was added to the solution, and the samples were incubated at 4°C for 2 h. The beads were washed with RIPA buffer (1% Triton X-100, 0.1% SDS, 0.1% DOC, 1 mM EDTA [pH 8.0], and 50 mM Tris-HCl [pH 8.0]) with 150 mM NaCl, RIPA buffer with 500 mM NaCl, LiCl wash solution (0.5% DOC, 0.5% NP-40, 1 mM EDTA [pH 8.0], 250 mM LiCl, and 10 mM Tris-HCl [pH 8.0]), and Tris-EDTA (TE).

    Article Title: Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages
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    Santa Cruz Biotechnology p stat1
    Our proposed model of nsDNA-induced APOL1 expression through engagement of the cGAS/IFI16-STING pathway in human immortalized AB8/13 podocytes. Binding of cytosolic nsDNA by cGAS and IFI16 activates STING, which subsequently activates TBK1. Activated TBK1 phosphorylates IRF3, which promotes transcription of APOL1 and IFNβ . IFNβ released from the cells (or exogenous IFNβ) binds to IFNAR. IFNAR-associated JAK1 and Tyk2 kinases then phosphorylate <t>STAT1,</t> which promotes transcription of APOL1 and IFI16 . A putative IFI16-mediated activation of STING is indicated by a dashed arrow. A potential cooperation between cGAS and IFI16 is indicated by a double-headed arrow. Deficient STING phosphorylation observed in cGAS- or IFI16-knockdown cells (Fig. 6 ) suggests that nsDNA-induced APOL1 expression may be mediated by a phospho-STING-independent pathway, marked by a green arrow. A dual JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) suppresses STAT1 activation and thereby inhibits IFI16 expression and STAT1-mediated APOL1 expression. Since IFNAR-mediated signaling involves JAK1 but not JAK2, our model only depicts the inhibition of JAK1 by Ruxo. Ruxo does not affect IRF3-mediated APOL1 expression.
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    Our proposed model of nsDNA-induced APOL1 expression through engagement of the cGAS/IFI16-STING pathway in human immortalized AB8/13 podocytes. Binding of cytosolic nsDNA by cGAS and IFI16 activates STING, which subsequently activates TBK1. Activated TBK1 phosphorylates IRF3, which promotes transcription of APOL1 and IFNβ . IFNβ released from the cells (or exogenous IFNβ) binds to IFNAR. IFNAR-associated JAK1 and Tyk2 kinases then phosphorylate STAT1, which promotes transcription of APOL1 and IFI16 . A putative IFI16-mediated activation of STING is indicated by a dashed arrow. A potential cooperation between cGAS and IFI16 is indicated by a double-headed arrow. Deficient STING phosphorylation observed in cGAS- or IFI16-knockdown cells (Fig. 6 ) suggests that nsDNA-induced APOL1 expression may be mediated by a phospho-STING-independent pathway, marked by a green arrow. A dual JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) suppresses STAT1 activation and thereby inhibits IFI16 expression and STAT1-mediated APOL1 expression. Since IFNAR-mediated signaling involves JAK1 but not JAK2, our model only depicts the inhibition of JAK1 by Ruxo. Ruxo does not affect IRF3-mediated APOL1 expression.

    Journal: Scientific Reports

    Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

    doi: 10.1038/s41598-019-51998-w

    Figure Lengend Snippet: Our proposed model of nsDNA-induced APOL1 expression through engagement of the cGAS/IFI16-STING pathway in human immortalized AB8/13 podocytes. Binding of cytosolic nsDNA by cGAS and IFI16 activates STING, which subsequently activates TBK1. Activated TBK1 phosphorylates IRF3, which promotes transcription of APOL1 and IFNβ . IFNβ released from the cells (or exogenous IFNβ) binds to IFNAR. IFNAR-associated JAK1 and Tyk2 kinases then phosphorylate STAT1, which promotes transcription of APOL1 and IFI16 . A putative IFI16-mediated activation of STING is indicated by a dashed arrow. A potential cooperation between cGAS and IFI16 is indicated by a double-headed arrow. Deficient STING phosphorylation observed in cGAS- or IFI16-knockdown cells (Fig. 6 ) suggests that nsDNA-induced APOL1 expression may be mediated by a phospho-STING-independent pathway, marked by a green arrow. A dual JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) suppresses STAT1 activation and thereby inhibits IFI16 expression and STAT1-mediated APOL1 expression. Since IFNAR-mediated signaling involves JAK1 but not JAK2, our model only depicts the inhibition of JAK1 by Ruxo. Ruxo does not affect IRF3-mediated APOL1 expression.

    Article Snippet: The following antibodies were used: APOL1 (Sigma, HPA018885, 1:5000), cGAS (Cell Signaling, D1D3G, 1:1000), IFI16 (Santa Cruz, sc-8023, 1:200), STING (Cell Signaling, D2P2F, 1:1000), P-STING (S366, Cell Signaling, D7C3S, 1:1000), TBK1 (Cell Signaling, D1B4, 1:500), P-TBK1 (S172, Cell Signaling, D52C2, 1:500), IRF3 (Cell Signaling, D6I4C, 1:1000), P-IRF3 (S386, Sigma, ABE501, 1:1000), STAT1 (Santa Cruz, sc-464, 1:500), P-STAT1 (Y701, Santa Cruz, sc-136229, 1:500), and GAPDH (Santa Cruz, sc-25778, 1:15000).

    Techniques: Expressing, Binding Assay, Activation Assay, Inhibition

    nsDNA-induced APOL1 expression is partly attenuated by JAK1/JAK2 inhibitor Ruxolitinib. ( a ) Ruxolitinib (Ruxo) inhibited IFNβ-mediated phosphorylation of STAT1. Sets of AB8/13 podocytes were treated with DMSO (solvent) only (Control), treated for 2 h with 5 μM Ruxo, treated for 15 min with 10 ng ml −1 IFNβ, or pretreated with Ruxo for 2 h followed by IFNβ stimulation for 15 min (Ruxo/IFNβ). The blot images were obtained from different gels. The blot probed for P-STAT1 was re-probed for GAPDH. ( b , c ) AB8/13 podocytes were treated with Ruxo and IFNβ as indicated and subsequently transfected with 1 μg ml −1 nsDNA for 2 h ( b ) or 18 h ( c ). The IFI16/GAPDH and APOL1/GAPDH ratios in unstimulated cells (Control) were both set as 1.0. The blot images in ( b ) were obtained from different gels. The blot probed for P-IRF3 was re-probed for P-STING. Other blot images were cropped from individually probed blots. The blot images in ( c ) were obtained from different gels. The blot probed for IFI16 was re-probed for cGAS. Other blot images were cropped from individually probed blots. Full images of all blots are shown in Supplementary Fig. S7 . ( d , e ) Ruxo abolished expression of APOL1 ( d ) and IFI16 mRNA ( e ) induced by exogenous IFNβ. ( f , g ) Ruxo partially inhibited nsDNA-induced APOL1 mRNA expression ( f ) but abolished nsDNA-induced IFI16 mRNA expression ( g ). Expression of APOL1 and IFNβ mRNA was analyzed by qRT-PCR 18 h after transfection with 1 μg ml −1 nsDNA. mRNA expression was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

    Journal: Scientific Reports

    Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

    doi: 10.1038/s41598-019-51998-w

    Figure Lengend Snippet: nsDNA-induced APOL1 expression is partly attenuated by JAK1/JAK2 inhibitor Ruxolitinib. ( a ) Ruxolitinib (Ruxo) inhibited IFNβ-mediated phosphorylation of STAT1. Sets of AB8/13 podocytes were treated with DMSO (solvent) only (Control), treated for 2 h with 5 μM Ruxo, treated for 15 min with 10 ng ml −1 IFNβ, or pretreated with Ruxo for 2 h followed by IFNβ stimulation for 15 min (Ruxo/IFNβ). The blot images were obtained from different gels. The blot probed for P-STAT1 was re-probed for GAPDH. ( b , c ) AB8/13 podocytes were treated with Ruxo and IFNβ as indicated and subsequently transfected with 1 μg ml −1 nsDNA for 2 h ( b ) or 18 h ( c ). The IFI16/GAPDH and APOL1/GAPDH ratios in unstimulated cells (Control) were both set as 1.0. The blot images in ( b ) were obtained from different gels. The blot probed for P-IRF3 was re-probed for P-STING. Other blot images were cropped from individually probed blots. The blot images in ( c ) were obtained from different gels. The blot probed for IFI16 was re-probed for cGAS. Other blot images were cropped from individually probed blots. Full images of all blots are shown in Supplementary Fig. S7 . ( d , e ) Ruxo abolished expression of APOL1 ( d ) and IFI16 mRNA ( e ) induced by exogenous IFNβ. ( f , g ) Ruxo partially inhibited nsDNA-induced APOL1 mRNA expression ( f ) but abolished nsDNA-induced IFI16 mRNA expression ( g ). Expression of APOL1 and IFNβ mRNA was analyzed by qRT-PCR 18 h after transfection with 1 μg ml −1 nsDNA. mRNA expression was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

    Article Snippet: The following antibodies were used: APOL1 (Sigma, HPA018885, 1:5000), cGAS (Cell Signaling, D1D3G, 1:1000), IFI16 (Santa Cruz, sc-8023, 1:200), STING (Cell Signaling, D2P2F, 1:1000), P-STING (S366, Cell Signaling, D7C3S, 1:1000), TBK1 (Cell Signaling, D1B4, 1:500), P-TBK1 (S172, Cell Signaling, D52C2, 1:500), IRF3 (Cell Signaling, D6I4C, 1:1000), P-IRF3 (S386, Sigma, ABE501, 1:1000), STAT1 (Santa Cruz, sc-464, 1:500), P-STAT1 (Y701, Santa Cruz, sc-136229, 1:500), and GAPDH (Santa Cruz, sc-25778, 1:15000).

    Techniques: Expressing, Transfection, Quantitative RT-PCR

    STAT1 binds to T FH cell signature genes

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

    Article Title: Type I Interferon induces binding of STAT1 to Bcl6: Divergent Roles of STAT-family transcription factors in the TFH cell genetic program

    doi: 10.4049/jimmunol.1300675

    Figure Lengend Snippet: STAT1 binds to T FH cell signature genes

    Article Snippet: Chromatin immunoprecipitation (ChIP)�sequencing (seq) experiments and data processing were performed as previously described ( , ) using anti-STAT1 antibody (Santa Cruz: sc-592).

    Techniques:

    STAT1, but not STAT3 or STAT4 is required for type I IFN mediated induction of key T FH cell molecules

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

    Article Title: Type I Interferon induces binding of STAT1 to Bcl6: Divergent Roles of STAT-family transcription factors in the TFH cell genetic program

    doi: 10.4049/jimmunol.1300675

    Figure Lengend Snippet: STAT1, but not STAT3 or STAT4 is required for type I IFN mediated induction of key T FH cell molecules

    Article Snippet: Chromatin immunoprecipitation (ChIP)�sequencing (seq) experiments and data processing were performed as previously described ( , ) using anti-STAT1 antibody (Santa Cruz: sc-592).

    Techniques:

    Effect of TAK165 and ATRA on STAT1 activation. ( a ) Western blot analysis of p-STAT1 (Tyr701 and Ser727) and STAT1 in the HL60 and NB4 cells. The cells were treated with TAK165 (100 nM for HL60, 50 nM for NB4) and ATRA (1 μM for HL60, 2 nM for NB4) for 3 days. ( b ) The relative phosphorylation level of STAT1 at Tyr701 and Ser727 in the HL60 cells, were evaluated by p-STAT1 (Tyr701 or Ser727)/STAT1. ( c ) STAT1 expression in the NB4-scrambleand NB4-shRNA-STAT1 cells was measured by western blot. ( d ) CD11b expression in the NB4-scrambleand NB4-shRNA-STAT1 cells. The cells were treated with 50 nM TAK165 and 2 nM ATRA for 3 days. The data presented are the mean ± SD. *** p

    Journal: Scientific Reports

    Article Title: The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARα/STAT1 axis

    doi: 10.1038/srep24589

    Figure Lengend Snippet: Effect of TAK165 and ATRA on STAT1 activation. ( a ) Western blot analysis of p-STAT1 (Tyr701 and Ser727) and STAT1 in the HL60 and NB4 cells. The cells were treated with TAK165 (100 nM for HL60, 50 nM for NB4) and ATRA (1 μM for HL60, 2 nM for NB4) for 3 days. ( b ) The relative phosphorylation level of STAT1 at Tyr701 and Ser727 in the HL60 cells, were evaluated by p-STAT1 (Tyr701 or Ser727)/STAT1. ( c ) STAT1 expression in the NB4-scrambleand NB4-shRNA-STAT1 cells was measured by western blot. ( d ) CD11b expression in the NB4-scrambleand NB4-shRNA-STAT1 cells. The cells were treated with 50 nM TAK165 and 2 nM ATRA for 3 days. The data presented are the mean ± SD. *** p

    Article Snippet: Antibodies against c-Myc (3G32), p-STAT1 (Tyr701), ERK1 (K-23), JNK (FL), MEK1/2, p38 (H-147), p-MEK-1 (Thr291), p-ERK1/2 (Thr204), and GAPDH (FL-335) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Western Blot, Expressing, shRNA

    Loss of Y372 phosphorylation reduces Jak2-mediated STAT1 phosphorylation

    Journal: Cellular signalling

    Article Title: PHOSPHORYLATION OF Y372 IS CRITICAL FOR JAK2 TYROSINE KINASE ACTIVATION

    doi: 10.1016/j.cellsig.2011.06.015

    Figure Lengend Snippet: Loss of Y372 phosphorylation reduces Jak2-mediated STAT1 phosphorylation

    Article Snippet: To demonstrate equal STAT1 precipitation across all samples, the membrane was stripped and re-blotted with anti-STAT1 antibody ( ).

    Techniques:

    The Jak2-Y372F mutant has an impaired ability to co-associate with STAT1

    Journal: Cellular signalling

    Article Title: PHOSPHORYLATION OF Y372 IS CRITICAL FOR JAK2 TYROSINE KINASE ACTIVATION

    doi: 10.1016/j.cellsig.2011.06.015

    Figure Lengend Snippet: The Jak2-Y372F mutant has an impaired ability to co-associate with STAT1

    Article Snippet: To demonstrate equal STAT1 precipitation across all samples, the membrane was stripped and re-blotted with anti-STAT1 antibody ( ).

    Techniques: Mutagenesis