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  • 99
    Name:
    Stat1 Antibody
    Description:
    The Stat1 transcription factor is activated in response to a large number of ligands 1 and is essential for responsiveness to IFN α and IFN γ 2 3 Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization nuclear translocation and DNA binding 4 Stat1 protein exists as a pair of isoforms Stat1α 91 kDa and the splice variant Stat1β 84 kDa In most cells both isoforms are activated by IFN α but only Stat1α is activated by IFN γ The inappropriate activation of Stat1 occurs in many tumors 5 In addition to tyrosine phosphorylation Stat1 is also phosphorylated at Ser727 through a p38 mitogen activated protein kinase MAPK dependent pathway in response to IFN α and other cellular stresses 6 Serine phosphorylation may be required for the maximal induction of Stat1 mediated gene activation
    Catalog Number:
    9172
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human Stat1. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti stat1
    Biological interferon production assay. A549 cells were infected at an MOI of 1 with BUNV, rBUNdelNSs2, rOROV, rOROVdelNSm, rOROV2080S, rOROVdelNSs, or rOROV246NSs or mock infected. Supernatant was harvested at 24 h p.i., and cell extracts were separated by SDS-PAGE. (A) UV-inactivated supernatant was used to pretreat A549-N pro cells prior to infection with EMCV. At 3 days p.i., cells were fixed and stained with crystal violet. (B) Graph calculated from panel A, presenting relative IFN units expressed as 2N where N is the number of 2-fold dilutions that offered protection. (C) Cells extracts were probed for OROV N, <t>STAT1,</t> pSTAT1, and MxA. Tubulin was probed as a loading control.
    The Stat1 transcription factor is activated in response to a large number of ligands 1 and is essential for responsiveness to IFN α and IFN γ 2 3 Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization nuclear translocation and DNA binding 4 Stat1 protein exists as a pair of isoforms Stat1α 91 kDa and the splice variant Stat1β 84 kDa In most cells both isoforms are activated by IFN α but only Stat1α is activated by IFN γ The inappropriate activation of Stat1 occurs in many tumors 5 In addition to tyrosine phosphorylation Stat1 is also phosphorylated at Ser727 through a p38 mitogen activated protein kinase MAPK dependent pathway in response to IFN α and other cellular stresses 6 Serine phosphorylation may be required for the maximal induction of Stat1 mediated gene activation
    https://www.bioz.com/result/anti stat1/product/Cell Signaling Technology Inc
    Average 99 stars, based on 173 article reviews
    Price from $9.99 to $1999.99
    anti stat1 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs"

    Article Title: Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs

    Journal: Journal of Virology

    doi: 10.1128/JVI.02849-15

    Biological interferon production assay. A549 cells were infected at an MOI of 1 with BUNV, rBUNdelNSs2, rOROV, rOROVdelNSm, rOROV2080S, rOROVdelNSs, or rOROV246NSs or mock infected. Supernatant was harvested at 24 h p.i., and cell extracts were separated by SDS-PAGE. (A) UV-inactivated supernatant was used to pretreat A549-N pro cells prior to infection with EMCV. At 3 days p.i., cells were fixed and stained with crystal violet. (B) Graph calculated from panel A, presenting relative IFN units expressed as 2N where N is the number of 2-fold dilutions that offered protection. (C) Cells extracts were probed for OROV N, STAT1, pSTAT1, and MxA. Tubulin was probed as a loading control.
    Figure Legend Snippet: Biological interferon production assay. A549 cells were infected at an MOI of 1 with BUNV, rBUNdelNSs2, rOROV, rOROVdelNSm, rOROV2080S, rOROVdelNSs, or rOROV246NSs or mock infected. Supernatant was harvested at 24 h p.i., and cell extracts were separated by SDS-PAGE. (A) UV-inactivated supernatant was used to pretreat A549-N pro cells prior to infection with EMCV. At 3 days p.i., cells were fixed and stained with crystal violet. (B) Graph calculated from panel A, presenting relative IFN units expressed as 2N where N is the number of 2-fold dilutions that offered protection. (C) Cells extracts were probed for OROV N, STAT1, pSTAT1, and MxA. Tubulin was probed as a loading control.

    Techniques Used: Infection, SDS Page, Staining

    2) Product Images from "Guanylate binding protein 1 is a novel effector of EGFR-driven invasion in glioblastoma"

    Article Title: Guanylate binding protein 1 is a novel effector of EGFR-driven invasion in glioblastoma

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20111102

    Stat1 is not required for EGFR-mediated GBP1 expression. (A) U87-EGFR cells were pretreated with 20 µM SB203580 for 1 h and then exposed to 100 ng/ml EGF or 100 ng/ml IFN-γ for 30 min before Western blot analysis. Total p38 is shown as a loading control. (B and C) U87-EGFR cells were transfected with Stat1 siRNA (si-stat1) or the control siRNA (si-Luc) before 20 ng/ml EGF (B) or 20 ng/ml IFN-γ treatment (C) for 24 h, and GBP1 expression was analyzed by Western blot. Data are representative of two independent experiments. (D) Stat1-null U3a cells are derived from parental 2fTGH cells. U3a-S1 cells are U3a cells reconstituted for Stat1. The cells were treated with 0, 5, 10, 20, 50, or 100 ng/ml IFN-γ for 24 h before Western blot analysis. (E) U3a and U3a-s1 cells transduced with EGFR were treated with 20 ng/ml EGF for 24 h before Western blot analysis. Data are representative of two independent experiments.
    Figure Legend Snippet: Stat1 is not required for EGFR-mediated GBP1 expression. (A) U87-EGFR cells were pretreated with 20 µM SB203580 for 1 h and then exposed to 100 ng/ml EGF or 100 ng/ml IFN-γ for 30 min before Western blot analysis. Total p38 is shown as a loading control. (B and C) U87-EGFR cells were transfected with Stat1 siRNA (si-stat1) or the control siRNA (si-Luc) before 20 ng/ml EGF (B) or 20 ng/ml IFN-γ treatment (C) for 24 h, and GBP1 expression was analyzed by Western blot. Data are representative of two independent experiments. (D) Stat1-null U3a cells are derived from parental 2fTGH cells. U3a-S1 cells are U3a cells reconstituted for Stat1. The cells were treated with 0, 5, 10, 20, 50, or 100 ng/ml IFN-γ for 24 h before Western blot analysis. (E) U3a and U3a-s1 cells transduced with EGFR were treated with 20 ng/ml EGF for 24 h before Western blot analysis. Data are representative of two independent experiments.

    Techniques Used: Expressing, Western Blot, Transfection, Derivative Assay, Transduction

    3) Product Images from "Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus"

    Article Title: Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092771

    Effect of recombinant IL-21 on expression of CD56, STAT proteins, and IFN-γ in NK-92 cells. NK-92 cells were cultured in the presence or absence of recombinant IL-21 (0.2 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were incubated with or without recombinant IL-21 (0.2 ng/mL) for 6 h and subsequently cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells for 12 h. Cell lysates from NK-92 cells only were assessed for the expressions of STAT1 and STAT5 proteins. β-actin was served as the loading control. IFN-γ production (pg/mL) in culture supernatants was determined by ELISA. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot; ( D ) Relative band intensity of STAT1 and STAT5 proteins compared with the loading control from at least three independent experiments; ( E ) IFN-γ production (pg/mL).
    Figure Legend Snippet: Effect of recombinant IL-21 on expression of CD56, STAT proteins, and IFN-γ in NK-92 cells. NK-92 cells were cultured in the presence or absence of recombinant IL-21 (0.2 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were incubated with or without recombinant IL-21 (0.2 ng/mL) for 6 h and subsequently cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells for 12 h. Cell lysates from NK-92 cells only were assessed for the expressions of STAT1 and STAT5 proteins. β-actin was served as the loading control. IFN-γ production (pg/mL) in culture supernatants was determined by ELISA. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot; ( D ) Relative band intensity of STAT1 and STAT5 proteins compared with the loading control from at least three independent experiments; ( E ) IFN-γ production (pg/mL).

    Techniques Used: Recombinant, Expressing, Cell Culture, Staining, FACS, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

    Effect of anti-IL-10 on expression of CD56, and STAT proteins in NK-92 cells. NK-92 cells were cultured in the presence or absence of anti-IL-10 (0.1 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells in the presence or absence of anti-IL-10 for 6 h. Cell lysates from NK-92 cells only were assessed for the expressions of stat1 and stat5 proteins. β-actin was served as the loading control. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot. ( D ) Relative band intensity of STAT1 and STAT5 compared with the loading control from at least three independent experiments.
    Figure Legend Snippet: Effect of anti-IL-10 on expression of CD56, and STAT proteins in NK-92 cells. NK-92 cells were cultured in the presence or absence of anti-IL-10 (0.1 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells in the presence or absence of anti-IL-10 for 6 h. Cell lysates from NK-92 cells only were assessed for the expressions of stat1 and stat5 proteins. β-actin was served as the loading control. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot. ( D ) Relative band intensity of STAT1 and STAT5 compared with the loading control from at least three independent experiments.

    Techniques Used: Expressing, Cell Culture, Staining, FACS, Western Blot

    4) Product Images from "VSL#3 Probiotic Stimulates T-Cell Protein Tyrosine Phosphatase-Mediated Recovery of IFN-γ Induced Intestinal Epithelial Barrier Defects"

    Article Title: VSL#3 Probiotic Stimulates T-Cell Protein Tyrosine Phosphatase-Mediated Recovery of IFN-γ Induced Intestinal Epithelial Barrier Defects

    Journal: Inflammatory bowel diseases

    doi: 10.1097/MIB.0000000000000954

    VSL#3 attenuated STAT1 signaling in a TCPTP-dependent manner (A) T 84 monolayers were basolaterally treated with IFN-γ (1000 U/ml) for 24 hrs or VSL#3 (10 2 , 10 4 , 10 6 , 10 8 CFU/mL) apically for 9 hrs and STAT1 phosphorylation and total STAT1 levels were determined by subsequent Western blotting of cell lysates. While IFN- γ as expected increased phospho-STAT1, VSL#3 alone was without effect (representative blot of 3 experiments. (B,C) Densitometric analysis of Western blots from T 84 monolayers treated basolaterally with IFN-γ (1000 U/ml) for 24 hrs showed a significant increase in STAT1 phosphorylation relative to total STAT1 and β-actin (p
    Figure Legend Snippet: VSL#3 attenuated STAT1 signaling in a TCPTP-dependent manner (A) T 84 monolayers were basolaterally treated with IFN-γ (1000 U/ml) for 24 hrs or VSL#3 (10 2 , 10 4 , 10 6 , 10 8 CFU/mL) apically for 9 hrs and STAT1 phosphorylation and total STAT1 levels were determined by subsequent Western blotting of cell lysates. While IFN- γ as expected increased phospho-STAT1, VSL#3 alone was without effect (representative blot of 3 experiments. (B,C) Densitometric analysis of Western blots from T 84 monolayers treated basolaterally with IFN-γ (1000 U/ml) for 24 hrs showed a significant increase in STAT1 phosphorylation relative to total STAT1 and β-actin (p

    Techniques Used: Western Blot

    5) Product Images from "K12/SECTM1, an interferon-? regulated molecule, synergizes with CD28 to costimulate human T cell proliferation"

    Article Title: K12/SECTM1, an interferon-? regulated molecule, synergizes with CD28 to costimulate human T cell proliferation

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.1011498

    Induction of SECTM1 expression is dependent on STAT1 pathway.
    Figure Legend Snippet: Induction of SECTM1 expression is dependent on STAT1 pathway.

    Techniques Used: Expressing

    6) Product Images from "Correlation of STAT1 with Apoptosis and Cell-Cycle Markers in Esophageal Squamous Cell Carcinoma"

    Article Title: Correlation of STAT1 with Apoptosis and Cell-Cycle Markers in Esophageal Squamous Cell Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113928

    Immunohistochemistry for Bcl-2, Bcl-xL, survivin, p21 and cyclin D1. By immunohistochemistry applied to formalin-fixed paraffin-embedded tissues, all proteins were detectable in most ESCC tumors. The staining was predominantly cytoplasmic for Bcl-2, Bcl-xL and surviving and nuclear for p21 and cyclin D1. Based on the staining intensity, tumors in our cohort were categorized into positive or negative (a) Cytoplasmic negative expression of Bcl-2 (b) Cytoplasmic positive expression of Bcl-2 (c) Cytoplasmic negative expression of Bcl-xL (d) Cytoplasmic positive expression of Bcl-xL (e) Cytoplasmic negative expression of survivin (f) Cytoplasmic positive expression of survivin (g) Nuclear negative staining of p21(h) Nuclear positive staining of p21 (i) Nuclear negative staining of cyclin D1 (j) Nuclear positive staining of cyclin D1(k) Cytoplasmic negative expression of STAT1(l) Cytoplasmic positive expression of STAT1 (IHC stain, scale bar, 20 µm).
    Figure Legend Snippet: Immunohistochemistry for Bcl-2, Bcl-xL, survivin, p21 and cyclin D1. By immunohistochemistry applied to formalin-fixed paraffin-embedded tissues, all proteins were detectable in most ESCC tumors. The staining was predominantly cytoplasmic for Bcl-2, Bcl-xL and surviving and nuclear for p21 and cyclin D1. Based on the staining intensity, tumors in our cohort were categorized into positive or negative (a) Cytoplasmic negative expression of Bcl-2 (b) Cytoplasmic positive expression of Bcl-2 (c) Cytoplasmic negative expression of Bcl-xL (d) Cytoplasmic positive expression of Bcl-xL (e) Cytoplasmic negative expression of survivin (f) Cytoplasmic positive expression of survivin (g) Nuclear negative staining of p21(h) Nuclear positive staining of p21 (i) Nuclear negative staining of cyclin D1 (j) Nuclear positive staining of cyclin D1(k) Cytoplasmic negative expression of STAT1(l) Cytoplasmic positive expression of STAT1 (IHC stain, scale bar, 20 µm).

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

    Gene transfection of STAT1C upregulated apoptosis in ESCC cell lines. Using Western blot analysis, the gene transfection of STAT1C in SHEE, NE3, KYSE150 and KYSE510 cells was shown to be effective, since the levels of STAT1, phospho-STAT1 and FLAG were dramatically increased 2 days after STAT1C transfection. By western blots, gene transfection of STAT1C into these cell lines down-regulated several pro-apoptotic proteins (including BCL-2, BCL-xL, survivin), and promoted G1 cell-cycle arrest by decreasing cyclin D1 and increasing p21waf1. Cell lysates were collected 2 days after the gene transfection of STAT1C in all the cell lines.
    Figure Legend Snippet: Gene transfection of STAT1C upregulated apoptosis in ESCC cell lines. Using Western blot analysis, the gene transfection of STAT1C in SHEE, NE3, KYSE150 and KYSE510 cells was shown to be effective, since the levels of STAT1, phospho-STAT1 and FLAG were dramatically increased 2 days after STAT1C transfection. By western blots, gene transfection of STAT1C into these cell lines down-regulated several pro-apoptotic proteins (including BCL-2, BCL-xL, survivin), and promoted G1 cell-cycle arrest by decreasing cyclin D1 and increasing p21waf1. Cell lysates were collected 2 days after the gene transfection of STAT1C in all the cell lines.

    Techniques Used: Transfection, Western Blot

    7) Product Images from "Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3"

    Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007559

    Bclaf1 interacts with STAT1/STAT2/IRF9. (A) IB analysis of STAT1, STAT2, P-STAT1, P-STAT2 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 2h. IgG was used for control immunoprecipitation. (B) IB analysis of IRF9 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 4h. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1 truncations and Ha-tagged STAT1/STAT2IRF9 expression plasmids. (D) qRT-PCR analysis of IFIT1 mRNA levels in HEp-2 cells transfected with Flag-tagged EV, full-length Bclaf1 or its truncations expression plasmids followed by PBS or human IFNα (500U/mL) treatment for 3h. IB analyzed the expression of Bclaf1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the one-way ANOVA test. ***p
    Figure Legend Snippet: Bclaf1 interacts with STAT1/STAT2/IRF9. (A) IB analysis of STAT1, STAT2, P-STAT1, P-STAT2 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 2h. IgG was used for control immunoprecipitation. (B) IB analysis of IRF9 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 4h. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1 truncations and Ha-tagged STAT1/STAT2IRF9 expression plasmids. (D) qRT-PCR analysis of IFIT1 mRNA levels in HEp-2 cells transfected with Flag-tagged EV, full-length Bclaf1 or its truncations expression plasmids followed by PBS or human IFNα (500U/mL) treatment for 3h. IB analyzed the expression of Bclaf1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the one-way ANOVA test. ***p

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

    Bclaf1 binds with ISRE and promotes the association of ISGF3 with DNA. (A) ChIP analysis of STAT1/STAT2/IRF9 DNA-binding in promoters of IFIT1 and IFIT2 in HeLa WT and HeLa Bclaf1-KO cells simulated with PBS or human IFNα (500U/mL) for 1h. (B) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1. Unlabeled ISRE was used for control. (C) IB analysis of ISRE-binding Bclaf1. Unlabeled ISRE and Bio-GFP were used for control. (D) ChIP analysis of Bclaf1 DNA-binding in promoters of ISG15 , IFIT1 and IFIT2 in HeLa cells simulated with PBS or human IFNα (500U/mL) for 1h. An amplicon located in IFIT1 exon2 was also tested for control. (E) IB analysis of WT or mutated (1–3) Bio-ISRE pull-down Bclaf1.
    Figure Legend Snippet: Bclaf1 binds with ISRE and promotes the association of ISGF3 with DNA. (A) ChIP analysis of STAT1/STAT2/IRF9 DNA-binding in promoters of IFIT1 and IFIT2 in HeLa WT and HeLa Bclaf1-KO cells simulated with PBS or human IFNα (500U/mL) for 1h. (B) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1. Unlabeled ISRE was used for control. (C) IB analysis of ISRE-binding Bclaf1. Unlabeled ISRE and Bio-GFP were used for control. (D) ChIP analysis of Bclaf1 DNA-binding in promoters of ISG15 , IFIT1 and IFIT2 in HeLa cells simulated with PBS or human IFNα (500U/mL) for 1h. An amplicon located in IFIT1 exon2 was also tested for control. (E) IB analysis of WT or mutated (1–3) Bio-ISRE pull-down Bclaf1.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Amplification

    Loss of Bclaf1 attenuates IFNα-mediated STAT1/STAT2 phosphorylation. (A) IB analysis of phosphorylated(P)-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα (500U/mL) for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (B) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (C) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in cytoplasmic and nuclear extracts of HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. α-Tubulin and Histone H3 were used as the cytoplasmic and nuclear controls, respectively.
    Figure Legend Snippet: Loss of Bclaf1 attenuates IFNα-mediated STAT1/STAT2 phosphorylation. (A) IB analysis of phosphorylated(P)-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα (500U/mL) for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (B) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (C) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in cytoplasmic and nuclear extracts of HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. α-Tubulin and Histone H3 were used as the cytoplasmic and nuclear controls, respectively.

    Techniques Used: Transfection

    Bclaf1 interacts with ISGF3 mainly through STAT2. (A) GST pulldown analysis of the interaction between His-STAT1/STAT2/IRF9 and GST-Bclaf1 F2. (B) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged STAT1 or STAT2/IRF9 expression plasmids. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged IRF9 or STAT2/STAT1 expression plasmids. (D) IB analysis of STAT1, STAT2, IRF9 and Flag-Bclaf1 in nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line transfected with si-control or si-STAT2 followed by PBS or human IFNα (500U/mL) treatment for 3h. (E) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1.
    Figure Legend Snippet: Bclaf1 interacts with ISGF3 mainly through STAT2. (A) GST pulldown analysis of the interaction between His-STAT1/STAT2/IRF9 and GST-Bclaf1 F2. (B) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged STAT1 or STAT2/IRF9 expression plasmids. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged IRF9 or STAT2/STAT1 expression plasmids. (D) IB analysis of STAT1, STAT2, IRF9 and Flag-Bclaf1 in nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line transfected with si-control or si-STAT2 followed by PBS or human IFNα (500U/mL) treatment for 3h. (E) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1.

    Techniques Used: Transfection, Expressing

    8) Product Images from "NOVEL STAT1 MUTATION DISRUPTS SMALL UBIQUITIN-RELATED MODIFIER (SUMO) CONJUGATION CAUSING GAIN OF FUNCTION"

    Article Title: NOVEL STAT1 MUTATION DISRUPTS SMALL UBIQUITIN-RELATED MODIFIER (SUMO) CONJUGATION CAUSING GAIN OF FUNCTION

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2017.07.027

    E705V STAT1 failed to conjugate SUMO (A–D) Transfected COS-7 cells show SUMO1 and the slower migrating SUMO-STAT1 conjugate bands in the WT-STAT1, (A, B) L706S and (D) Y701C cells, but absent in (A–D) E705V, (A, B) K703R, (B, D) E705Q and (C, D) E705V+WT-STAT1; (D) Bands are absent following IP with the isotype antibody. Experiments were performed at least thrice.
    Figure Legend Snippet: E705V STAT1 failed to conjugate SUMO (A–D) Transfected COS-7 cells show SUMO1 and the slower migrating SUMO-STAT1 conjugate bands in the WT-STAT1, (A, B) L706S and (D) Y701C cells, but absent in (A–D) E705V, (A, B) K703R, (B, D) E705Q and (C, D) E705V+WT-STAT1; (D) Bands are absent following IP with the isotype antibody. Experiments were performed at least thrice.

    Techniques Used: Transfection

    Enhanced pSTAT1 in transfected cells STAT1-deficient cell lines transfected with WT, E705V and E705Q STAT1 constructs were stimulated or not (NS) with IFN-γ (1,000 IU/ml) and treated further with (A–C) staurosporine (n=5), or (D) pervanadate (one representative experiment out of two). Cell lysates were immunoblotted for pSTAT1, STAT1 and tubulin.
    Figure Legend Snippet: Enhanced pSTAT1 in transfected cells STAT1-deficient cell lines transfected with WT, E705V and E705Q STAT1 constructs were stimulated or not (NS) with IFN-γ (1,000 IU/ml) and treated further with (A–C) staurosporine (n=5), or (D) pervanadate (one representative experiment out of two). Cell lysates were immunoblotted for pSTAT1, STAT1 and tubulin.

    Techniques Used: Transfection, Construct

    Enhanced response in E705V transfected cells (A) DNA binding GAS activity, (B) transcriptional response and (C) gene expression were evaluated in cells transfected with WT and STAT1 mutants following IFN-γ stimulation. Data are mean fold (± SD) relative to WT non-stimulated (NS) from two independent experiments performed in triplicate. * p
    Figure Legend Snippet: Enhanced response in E705V transfected cells (A) DNA binding GAS activity, (B) transcriptional response and (C) gene expression were evaluated in cells transfected with WT and STAT1 mutants following IFN-γ stimulation. Data are mean fold (± SD) relative to WT non-stimulated (NS) from two independent experiments performed in triplicate. * p

    Techniques Used: Transfection, Binding Assay, Activity Assay, Expressing

    Accumulation of nuclear paracrystals Following IFN-γ stimulation (1,000 IU/mL), SUMO-free STAT1 accumulates in the nucleus as assembly particles. Transfected U3C cells (WT-STAT1-GFP; E705V-GFP) were stimulated or not for 1 h and fixed. Confocal microscopy shows accumulation of punctuate particles (arrows) in the E705V-STAT1 cells, which were absent in the WT-STAT1. One representative experiment out of four is presented.
    Figure Legend Snippet: Accumulation of nuclear paracrystals Following IFN-γ stimulation (1,000 IU/mL), SUMO-free STAT1 accumulates in the nucleus as assembly particles. Transfected U3C cells (WT-STAT1-GFP; E705V-GFP) were stimulated or not for 1 h and fixed. Confocal microscopy shows accumulation of punctuate particles (arrows) in the E705V-STAT1 cells, which were absent in the WT-STAT1. One representative experiment out of four is presented.

    Techniques Used: Transfection, Confocal Microscopy

    9) Product Images from "NOVEL STAT1 MUTATION DISRUPTS SMALL UBIQUITIN-RELATED MODIFIER (SUMO) CONJUGATION CAUSING GAIN OF FUNCTION"

    Article Title: NOVEL STAT1 MUTATION DISRUPTS SMALL UBIQUITIN-RELATED MODIFIER (SUMO) CONJUGATION CAUSING GAIN OF FUNCTION

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2017.07.027

    E705V STAT1 failed to conjugate SUMO (A–D) Transfected COS-7 cells show SUMO1 and the slower migrating SUMO-STAT1 conjugate bands in the WT-STAT1, (A, B) L706S and (D) Y701C cells, but absent in (A–D) E705V, (A, B) K703R, (B, D) E705Q and (C, D) E705V+WT-STAT1; (D) Bands are absent following IP with the isotype antibody. Experiments were performed at least thrice.
    Figure Legend Snippet: E705V STAT1 failed to conjugate SUMO (A–D) Transfected COS-7 cells show SUMO1 and the slower migrating SUMO-STAT1 conjugate bands in the WT-STAT1, (A, B) L706S and (D) Y701C cells, but absent in (A–D) E705V, (A, B) K703R, (B, D) E705Q and (C, D) E705V+WT-STAT1; (D) Bands are absent following IP with the isotype antibody. Experiments were performed at least thrice.

    Techniques Used: Transfection

    Enhanced pSTAT1 in transfected cells STAT1-deficient cell lines transfected with WT, E705V and E705Q STAT1 constructs were stimulated or not (NS) with IFN-γ (1,000 IU/ml) and treated further with (A–C) staurosporine (n=5), or (D) pervanadate (one representative experiment out of two). Cell lysates were immunoblotted for pSTAT1, STAT1 and tubulin.
    Figure Legend Snippet: Enhanced pSTAT1 in transfected cells STAT1-deficient cell lines transfected with WT, E705V and E705Q STAT1 constructs were stimulated or not (NS) with IFN-γ (1,000 IU/ml) and treated further with (A–C) staurosporine (n=5), or (D) pervanadate (one representative experiment out of two). Cell lysates were immunoblotted for pSTAT1, STAT1 and tubulin.

    Techniques Used: Transfection, Construct

    Enhanced response in E705V transfected cells (A) DNA binding GAS activity, (B) transcriptional response and (C) gene expression were evaluated in cells transfected with WT and STAT1 mutants following IFN-γ stimulation. Data are mean fold (± SD) relative to WT non-stimulated (NS) from two independent experiments performed in triplicate. * p
    Figure Legend Snippet: Enhanced response in E705V transfected cells (A) DNA binding GAS activity, (B) transcriptional response and (C) gene expression were evaluated in cells transfected with WT and STAT1 mutants following IFN-γ stimulation. Data are mean fold (± SD) relative to WT non-stimulated (NS) from two independent experiments performed in triplicate. * p

    Techniques Used: Transfection, Binding Assay, Activity Assay, Expressing

    Accumulation of nuclear paracrystals Following IFN-γ stimulation (1,000 IU/mL), SUMO-free STAT1 accumulates in the nucleus as assembly particles. Transfected U3C cells (WT-STAT1-GFP; E705V-GFP) were stimulated or not for 1 h and fixed. Confocal microscopy shows accumulation of punctuate particles (arrows) in the E705V-STAT1 cells, which were absent in the WT-STAT1. One representative experiment out of four is presented.
    Figure Legend Snippet: Accumulation of nuclear paracrystals Following IFN-γ stimulation (1,000 IU/mL), SUMO-free STAT1 accumulates in the nucleus as assembly particles. Transfected U3C cells (WT-STAT1-GFP; E705V-GFP) were stimulated or not for 1 h and fixed. Confocal microscopy shows accumulation of punctuate particles (arrows) in the E705V-STAT1 cells, which were absent in the WT-STAT1. One representative experiment out of four is presented.

    Techniques Used: Transfection, Confocal Microscopy

    10) Product Images from "Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment"

    Article Title: Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment

    Journal: Open Biology

    doi: 10.1098/rsob.160065

    STAT1 is a direct target of Bcl6 in C3H10T1/2 cells. ( a , b ) Knockdown of Bcl6 in C3H10T1/2 cells represses STAT1 expression, and overexpression of Bcl6 enhances STAT1 expression by qPCR and western blot, respectively. Values are mean ± s.d. ( n = 3). ** p
    Figure Legend Snippet: STAT1 is a direct target of Bcl6 in C3H10T1/2 cells. ( a , b ) Knockdown of Bcl6 in C3H10T1/2 cells represses STAT1 expression, and overexpression of Bcl6 enhances STAT1 expression by qPCR and western blot, respectively. Values are mean ± s.d. ( n = 3). ** p

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot

    STAT1 is required during adipogenesis and rescues the adipogenic competency in Bcl6 knockdown C3H10T1/2 cells. ( a , b ) The STAT1 mRNA and protein expression levels after transfected siNC or siSTAT1s after 36 h and 48 h, respectively. ( c ) Adipogenic phenotypes of C3H10T1/2 cells transiently transfected with synthetic siRNAs targeting STAT1 or NC siRNA after induced 8 days by MDII and stained with oil red-O, Scale bars, 50 µm. ( d ) The expression of adipocyte genes PPARγ2, aP2, LPL and adiponectin at day 8 was determined by qPCR. ( e ) The expression of adipocyte protein PPARγ and adiponectin at day 8 was determined by western blot. ( f ) Adipogenic phenotypes of C3H10T1/2 cells transiently transfected with pRNAT-shBcl6 or pRNAT-shNC, or cotransfected with pRNAT-shBcl6 and pCDNA3.1-STAT1 after induced by MDII, and stained with Oil red-O on day 8 of adipogenic differentiation, Scale bars, 50 µm. ( g ) The expression of adipocyte genes PPARγ2, aP2, LPL and adiponectin was detected by qPCR. ( h,i ) The mRNA and protein expression levels of STAT1 after transfected with Bcl6 domain truncation plasmids or empty vector after 36 h and 48 h, respectively. Values are represented as mean ± s.d. ( n = 3). * p
    Figure Legend Snippet: STAT1 is required during adipogenesis and rescues the adipogenic competency in Bcl6 knockdown C3H10T1/2 cells. ( a , b ) The STAT1 mRNA and protein expression levels after transfected siNC or siSTAT1s after 36 h and 48 h, respectively. ( c ) Adipogenic phenotypes of C3H10T1/2 cells transiently transfected with synthetic siRNAs targeting STAT1 or NC siRNA after induced 8 days by MDII and stained with oil red-O, Scale bars, 50 µm. ( d ) The expression of adipocyte genes PPARγ2, aP2, LPL and adiponectin at day 8 was determined by qPCR. ( e ) The expression of adipocyte protein PPARγ and adiponectin at day 8 was determined by western blot. ( f ) Adipogenic phenotypes of C3H10T1/2 cells transiently transfected with pRNAT-shBcl6 or pRNAT-shNC, or cotransfected with pRNAT-shBcl6 and pCDNA3.1-STAT1 after induced by MDII, and stained with Oil red-O on day 8 of adipogenic differentiation, Scale bars, 50 µm. ( g ) The expression of adipocyte genes PPARγ2, aP2, LPL and adiponectin was detected by qPCR. ( h,i ) The mRNA and protein expression levels of STAT1 after transfected with Bcl6 domain truncation plasmids or empty vector after 36 h and 48 h, respectively. Values are represented as mean ± s.d. ( n = 3). * p

    Techniques Used: Expressing, Transfection, Staining, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation

    11) Product Images from "A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-?B"

    Article Title: A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-?B

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-12-14

    Involvement of STAT1 expression level in STAT3-decoy ODN-induced cell death . Colon carcinoma SW 480 cells transduced either with control virus (black histograms) or with STAT1-specific shRNA (white histograms). A : Detection of STAT1 by western blotting of control cells (1), cells transduced with an empty virus (Ev) (lane 2), and with a STAT1-specific shRNA lentivirus (3). Below are shown the detection of STAT3, the NF-κB (p50 subunit) and actin in the same extracts. B : Detection of dead cells by trypan blue exclusion counting. Control non-treated (black histograms) and STAT1-shRNA-treated cells (white histograms) were either not treated or transfected with STAT3-decoy ODN (ST3dODN) (48 h), mutated STAT3-decoy ODN (mtdODN) (48 h) or IFNγ (100 ng/ml, 48 h). Data are expressed in % of dead cells.
    Figure Legend Snippet: Involvement of STAT1 expression level in STAT3-decoy ODN-induced cell death . Colon carcinoma SW 480 cells transduced either with control virus (black histograms) or with STAT1-specific shRNA (white histograms). A : Detection of STAT1 by western blotting of control cells (1), cells transduced with an empty virus (Ev) (lane 2), and with a STAT1-specific shRNA lentivirus (3). Below are shown the detection of STAT3, the NF-κB (p50 subunit) and actin in the same extracts. B : Detection of dead cells by trypan blue exclusion counting. Control non-treated (black histograms) and STAT1-shRNA-treated cells (white histograms) were either not treated or transfected with STAT3-decoy ODN (ST3dODN) (48 h), mutated STAT3-decoy ODN (mtdODN) (48 h) or IFNγ (100 ng/ml, 48 h). Data are expressed in % of dead cells.

    Techniques Used: Expressing, shRNA, Western Blot, Transduction, Transfection

    12) Product Images from "Specific engagement of TLR4 or TLR3 does not lead to IFN-?-mediated innate signal amplification and STAT1 phosphorylation in resident murine alveolar macrophages 1"

    Article Title: Specific engagement of TLR4 or TLR3 does not lead to IFN-?-mediated innate signal amplification and STAT1 phosphorylation in resident murine alveolar macrophages 1

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

    doi:

    Murine AMø do not phosphorylate STAT1 in response to TLR engagement, but do so in response to exogenous IFNs. A, B. TLR engagement. Resident PMø (panels A) or AMø (panels B) were incubated for 2.5 or 5h in the presence of medium
    Figure Legend Snippet: Murine AMø do not phosphorylate STAT1 in response to TLR engagement, but do so in response to exogenous IFNs. A, B. TLR engagement. Resident PMø (panels A) or AMø (panels B) were incubated for 2.5 or 5h in the presence of medium

    Techniques Used: Incubation

    13) Product Images from "K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo"

    Article Title: K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2016.51

    RIPK1 K45A impacts JNK activation, STAT1 phosphorylation at position S727 and chemokine production by macrophages. WT or RIPK1 K45A macrophages were treated as indicated in the panels and tested for STAT1 and JNK phosphorylation ( a , c , e ) by western blotting analysis. ( b , d , f ) Densitometric analysis of the representative western blottings. Chemokine production by macrophages was measured ( g ) at various time intervals or ( h ) at 6 h poststimulation of cells. ( i ) mRNA levels of the chemokines measured in panels ( g and h ) were analyzed under same conditions. The mRNA levels were normalized to rpp30. ANOVA with Bonferroni post-test was used to measure statistical significance, ** P
    Figure Legend Snippet: RIPK1 K45A impacts JNK activation, STAT1 phosphorylation at position S727 and chemokine production by macrophages. WT or RIPK1 K45A macrophages were treated as indicated in the panels and tested for STAT1 and JNK phosphorylation ( a , c , e ) by western blotting analysis. ( b , d , f ) Densitometric analysis of the representative western blottings. Chemokine production by macrophages was measured ( g ) at various time intervals or ( h ) at 6 h poststimulation of cells. ( i ) mRNA levels of the chemokines measured in panels ( g and h ) were analyzed under same conditions. The mRNA levels were normalized to rpp30. ANOVA with Bonferroni post-test was used to measure statistical significance, ** P

    Techniques Used: Activation Assay, Western Blot

    14) Product Images from "Testosterone suppresses uropathogenic Escherichia coli invasion and colonization within prostate cells and inhibits inflammatory responses through JAK/STAT-1 signaling pathway"

    Article Title: Testosterone suppresses uropathogenic Escherichia coli invasion and colonization within prostate cells and inhibits inflammatory responses through JAK/STAT-1 signaling pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0180244

    Testosterone downregulated the mRNA levels of JAK/STAT1 signaling pathway components in LPS-stimulated inflammation of prostate cells. Total RNA isolated from treated cells was analyzed using qPCR. The mRNA levels of JAK1 , JAK2 , STAT1 , IFNG , IL-6 , IL1B , and IL-8 were detected in LPS-stimulated prostate cells treated with different doses of testosterone (T) (10, 20, and 40 μg/mL) for 24 h. Testosterone alone (10, 20, and 40 μg/mL) treatments for 24 h were performed as controls. Data from 3 separate experiments were expressed as mean ± SD when normalized to the expression of the internal control gene for the 18S ribosomal RNA. * p
    Figure Legend Snippet: Testosterone downregulated the mRNA levels of JAK/STAT1 signaling pathway components in LPS-stimulated inflammation of prostate cells. Total RNA isolated from treated cells was analyzed using qPCR. The mRNA levels of JAK1 , JAK2 , STAT1 , IFNG , IL-6 , IL1B , and IL-8 were detected in LPS-stimulated prostate cells treated with different doses of testosterone (T) (10, 20, and 40 μg/mL) for 24 h. Testosterone alone (10, 20, and 40 μg/mL) treatments for 24 h were performed as controls. Data from 3 separate experiments were expressed as mean ± SD when normalized to the expression of the internal control gene for the 18S ribosomal RNA. * p

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

    Effects of JAK and STAT1 inhibitor on testosterone-mediated suppression of UPEC colonization in PZ-HPV-7 cells. PZ-HPV-7 cells were pretreated with 40 μg/mL testosterone (T) and 50 μM JAK inhibitor or 50 μM STAT1 inhibitor for 24 h, followed by UPEC infection (MOI; 100) as described above. Cells pretreated with 50 μM JAK inhibitor or STAT1 inhibitor alone for 24 h followed by UPEC infection were used as controls. Twenty-four hours post-infection, all infected cells were lysed and plated on LB agar (A, B) or detected using flow cytometry (C) for analyzing UPEC colonization. Data are expressed as the mean ± SD from 3 separate experiments. Colony forming units (CFU) were acquired after plating ten-fold dilutions of infected cells post-lysis. Flow cytometry data are shown as dot-blots for all analyzed samples, and one representative per experiment is shown. Numbers in the right lower quadrant show the frequency of GFP-expressing cells in the quadrant of total cells. * p
    Figure Legend Snippet: Effects of JAK and STAT1 inhibitor on testosterone-mediated suppression of UPEC colonization in PZ-HPV-7 cells. PZ-HPV-7 cells were pretreated with 40 μg/mL testosterone (T) and 50 μM JAK inhibitor or 50 μM STAT1 inhibitor for 24 h, followed by UPEC infection (MOI; 100) as described above. Cells pretreated with 50 μM JAK inhibitor or STAT1 inhibitor alone for 24 h followed by UPEC infection were used as controls. Twenty-four hours post-infection, all infected cells were lysed and plated on LB agar (A, B) or detected using flow cytometry (C) for analyzing UPEC colonization. Data are expressed as the mean ± SD from 3 separate experiments. Colony forming units (CFU) were acquired after plating ten-fold dilutions of infected cells post-lysis. Flow cytometry data are shown as dot-blots for all analyzed samples, and one representative per experiment is shown. Numbers in the right lower quadrant show the frequency of GFP-expressing cells in the quadrant of total cells. * p

    Techniques Used: Infection, Flow Cytometry, Cytometry, Lysis, Expressing

    Testosterone downregulated the protein levels of JAK/STAT1 signaling pathway components in LPS-stimulated inflammation of prostate cells. PZ-HPV-7 cells were treated with different doses of testosterone (T) (10, 20, and 40 μg/mL) and stimulated with LPS (5 μg/mL) for 24 h, followed by collection of the total protein of all the treatment groups. The protein levels of JAK1, JAK2, STAT1, IFN-γ, IL-6, and IL-1β were analyzed using western blotting. All data were normalized to that of the internal reference β-actin. Results were assessed using densitometry and quantified using the ImageJ software (NIH). The results are shown as the mean ± SD from three independent experiments. * p
    Figure Legend Snippet: Testosterone downregulated the protein levels of JAK/STAT1 signaling pathway components in LPS-stimulated inflammation of prostate cells. PZ-HPV-7 cells were treated with different doses of testosterone (T) (10, 20, and 40 μg/mL) and stimulated with LPS (5 μg/mL) for 24 h, followed by collection of the total protein of all the treatment groups. The protein levels of JAK1, JAK2, STAT1, IFN-γ, IL-6, and IL-1β were analyzed using western blotting. All data were normalized to that of the internal reference β-actin. Results were assessed using densitometry and quantified using the ImageJ software (NIH). The results are shown as the mean ± SD from three independent experiments. * p

    Techniques Used: Western Blot, Software

    15) Product Images from "Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells"

    Article Title: Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00268

    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P
    Figure Legend Snippet: DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Techniques Used: Expressing, Quantitative RT-PCR, Infection

    16) Product Images from "Interplay of hepatic and myeloid STAT3 in facilitating liver regeneration via tempering innate immunity"

    Article Title: Interplay of hepatic and myeloid STAT3 in facilitating liver regeneration via tempering innate immunity

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.23430

    An interplay of myeloid and hepatic STAT3 in preventing liver failure during liver regeneration via tempering innate immunity Myeloid STAT3 inhibits infiltration of innate immune cells, inhibits STAT1 activation in innate immune cells, and inhibits production of inflammatory cytokines such as TNF-α, IL-6, and IFN-γ; while hepatic STAT3 inhibits inflammatory signaling STAT1 in the liver and promotes hepatocyte survival.
    Figure Legend Snippet: An interplay of myeloid and hepatic STAT3 in preventing liver failure during liver regeneration via tempering innate immunity Myeloid STAT3 inhibits infiltration of innate immune cells, inhibits STAT1 activation in innate immune cells, and inhibits production of inflammatory cytokines such as TNF-α, IL-6, and IFN-γ; while hepatic STAT3 inhibits inflammatory signaling STAT1 in the liver and promotes hepatocyte survival.

    Techniques Used: Activation Assay

    Deletion of STAT1 diminishes inflammatory responses in STAT3 Mye−/− mice after PHx A . Western blot analyses of liver leukocytes to confirm STAT3 Mye−/− STAT1 −/− mice. Liver leukocytes were isolated 3 hrs post PHx. B . STAT3 Mye−/− STAT1 −/− mice had comparable liver regeneration as STAT3 Mye−/− mice after PHx. C . STAT3 Mye−/− STAT1 −/− mice showed reduced serum cytokines after PHx compared to STAT3 Mye−/− mice. Values represent means ± SD (n=5-9). * P
    Figure Legend Snippet: Deletion of STAT1 diminishes inflammatory responses in STAT3 Mye−/− mice after PHx A . Western blot analyses of liver leukocytes to confirm STAT3 Mye−/− STAT1 −/− mice. Liver leukocytes were isolated 3 hrs post PHx. B . STAT3 Mye−/− STAT1 −/− mice had comparable liver regeneration as STAT3 Mye−/− mice after PHx. C . STAT3 Mye−/− STAT1 −/− mice showed reduced serum cytokines after PHx compared to STAT3 Mye−/− mice. Values represent means ± SD (n=5-9). * P

    Techniques Used: Mouse Assay, Western Blot, Isolation

    STAT1 activation is enhanced the liver and leukocytes in STAT3 Hep−/− and STAT3 Mye−/− mice, respectively, and is enhanced in both the liver and leuokcytes in STAT3 Mye−/−Hep−/− mice after PHx. A, Western blot (upper panel) and RT-PCR (low panel) analyses of liver tissues post PHx. B, Western blot analyses of liver leukocytes 3 hrs post sham (s) and PHx (p). Representative Western blot photography was shown from 2 independent experiments with similar results. “–” represents control group without operation. WT: represent littermate controls.
    Figure Legend Snippet: STAT1 activation is enhanced the liver and leukocytes in STAT3 Hep−/− and STAT3 Mye−/− mice, respectively, and is enhanced in both the liver and leuokcytes in STAT3 Mye−/−Hep−/− mice after PHx. A, Western blot (upper panel) and RT-PCR (low panel) analyses of liver tissues post PHx. B, Western blot analyses of liver leukocytes 3 hrs post sham (s) and PHx (p). Representative Western blot photography was shown from 2 independent experiments with similar results. “–” represents control group without operation. WT: represent littermate controls.

    Techniques Used: Activation Assay, Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Deletion of STAT1 rescues liver failure in STAT3 Mye−/−Hep−/− mice after PHx A . Western blot analyses of liver tissues and leukocytes to confirm the STAT3 Mye−/−Hep−/− STAT1 −/− KO mice. B-D . Deletion of STAT1 increases the survival rate ( B ), restores liver regeneration and reduces apoptosis ( C ), and reduces the inflammatory response ( D ) in STAT3 Mye-Hep−/− mice after PHx. Values represent means ± SD (n=4-8). * P
    Figure Legend Snippet: Deletion of STAT1 rescues liver failure in STAT3 Mye−/−Hep−/− mice after PHx A . Western blot analyses of liver tissues and leukocytes to confirm the STAT3 Mye−/−Hep−/− STAT1 −/− KO mice. B-D . Deletion of STAT1 increases the survival rate ( B ), restores liver regeneration and reduces apoptosis ( C ), and reduces the inflammatory response ( D ) in STAT3 Mye-Hep−/− mice after PHx. Values represent means ± SD (n=4-8). * P

    Techniques Used: Mouse Assay, Western Blot

    Deletion of STAT1 restores liver regeneration in STAT3 Hep−/− mice and abolishes IFN-γ inhibition of hepatocyte proliferation A, Expression of STAT1 in the liver is enhanced in STAT3 Hep−/− mice after PHx. B, Western blot analyses of liver tissues to confirm STAT3 Hep−/− STAT1 −/− mice. C, STAT3 Hep−/− STAT1 −/− mice survived after PHx. D, STAT3 Hep−/− STAT1 −/− mice showed increased liver regeneration compared to STAT3 Hep−/− mice. E, Western blotting of primary mouse hepatocytes treated with IFN-γ. F, Primary hepatocytes were treated with IFN-γ for 48 hrs, cell proliferation was then determined by measuring [ 3 H] thymidine uptake. Values represent means ± SD (n=6-8). In panel D, ** P
    Figure Legend Snippet: Deletion of STAT1 restores liver regeneration in STAT3 Hep−/− mice and abolishes IFN-γ inhibition of hepatocyte proliferation A, Expression of STAT1 in the liver is enhanced in STAT3 Hep−/− mice after PHx. B, Western blot analyses of liver tissues to confirm STAT3 Hep−/− STAT1 −/− mice. C, STAT3 Hep−/− STAT1 −/− mice survived after PHx. D, STAT3 Hep−/− STAT1 −/− mice showed increased liver regeneration compared to STAT3 Hep−/− mice. E, Western blotting of primary mouse hepatocytes treated with IFN-γ. F, Primary hepatocytes were treated with IFN-γ for 48 hrs, cell proliferation was then determined by measuring [ 3 H] thymidine uptake. Values represent means ± SD (n=6-8). In panel D, ** P

    Techniques Used: Mouse Assay, Inhibition, Expressing, Western Blot

    17) Product Images from "CD1d1 intrinsic signaling in macrophages controls NLRP3 inflammasome expression during inflammation"

    Article Title: CD1d1 intrinsic signaling in macrophages controls NLRP3 inflammasome expression during inflammation

    Journal: Science Advances

    doi: 10.1126/sciadv.aaz7290

    iGb3/CD1d1 inactivates NF-κB through decreasing PRDX1 expression. Mouse BMDMs were treated with PBS, LPS (1 μg/ml; 6 hours), or LPS and iGb3 (5 μg/ml; 12 hours). ( A ) ChIP-qPCR analysis for RelA protein binding to the promoter regions of indicated genes. WT and CD1d1 −/− BMDMs were treated with LPS (1 μg/ml; 6 hours) or LPS and iGb3 (5 μg/ml; 12 hours), and ( B ) cell extracts were immunoblotted for the indicated molecules. ( C ) The expression of RelA in BMDMs was silenced by RNAi; after 48 hours, cells were treated with LPS (1 μg/ml; 6 hours), protein levels were analyzed by immunoblotting (left), and gene transcription was detected by qRT-PCR (right). ( D ) Immunoblotting analysis of indicated protein from BMDMs treated with the NF-κB inhibitor BAY 11-7082. ( E ) Mouse BMDMs were treated with PBS, LPS (1 μg/ml; 6 hours), or LPS and iGb3 (5 μg/ml; 12 hours); qRT-PCR analysis of Prdx-1 gene transcription. The expression of Prdx1 in RAW264.7 cells was silenced by RNAi; after 48 hours, cells were further treated with LPS (1 μg/ml; 6 hours). ( F ) Immunoblotting analysis of the indicated proteins. ( G ) qRT-PCR detected gene transcription of the indicated molecules. LPS-primed BMDMs were treated with ( H ) the AKT inhibitor MK2206, ( I ) the STAT1 inhibitor fludarabine ; immunoblotting analysis of the indicated molecules. ( J ) Mouse BMDMs were treated with PBS, LPS (1 μg/ml; 6 hours), or LPS and iGb3 (5 μg/ml; 12 hours); ChIP-qPCR analysis for STAT1 protein binding to the promoter regions of Prdx1 gene. ( K ) CD1d1 immunoprecipitation (IP) from BMDMs and immunoblotting of p-Ser. ( L ) CD1d1 −/− BMDMs were stably transfected with CD1d1 -vectors, CD1d1 S330A -vectors or pcDNA3.1(-) vectors (mock), cells were further treated with LPS and iGb3, and cell extracts were immunoblotted for IκBα and STAT1 phosphorylation. Error bar, SEM. * P
    Figure Legend Snippet: iGb3/CD1d1 inactivates NF-κB through decreasing PRDX1 expression. Mouse BMDMs were treated with PBS, LPS (1 μg/ml; 6 hours), or LPS and iGb3 (5 μg/ml; 12 hours). ( A ) ChIP-qPCR analysis for RelA protein binding to the promoter regions of indicated genes. WT and CD1d1 −/− BMDMs were treated with LPS (1 μg/ml; 6 hours) or LPS and iGb3 (5 μg/ml; 12 hours), and ( B ) cell extracts were immunoblotted for the indicated molecules. ( C ) The expression of RelA in BMDMs was silenced by RNAi; after 48 hours, cells were treated with LPS (1 μg/ml; 6 hours), protein levels were analyzed by immunoblotting (left), and gene transcription was detected by qRT-PCR (right). ( D ) Immunoblotting analysis of indicated protein from BMDMs treated with the NF-κB inhibitor BAY 11-7082. ( E ) Mouse BMDMs were treated with PBS, LPS (1 μg/ml; 6 hours), or LPS and iGb3 (5 μg/ml; 12 hours); qRT-PCR analysis of Prdx-1 gene transcription. The expression of Prdx1 in RAW264.7 cells was silenced by RNAi; after 48 hours, cells were further treated with LPS (1 μg/ml; 6 hours). ( F ) Immunoblotting analysis of the indicated proteins. ( G ) qRT-PCR detected gene transcription of the indicated molecules. LPS-primed BMDMs were treated with ( H ) the AKT inhibitor MK2206, ( I ) the STAT1 inhibitor fludarabine ; immunoblotting analysis of the indicated molecules. ( J ) Mouse BMDMs were treated with PBS, LPS (1 μg/ml; 6 hours), or LPS and iGb3 (5 μg/ml; 12 hours); ChIP-qPCR analysis for STAT1 protein binding to the promoter regions of Prdx1 gene. ( K ) CD1d1 immunoprecipitation (IP) from BMDMs and immunoblotting of p-Ser. ( L ) CD1d1 −/− BMDMs were stably transfected with CD1d1 -vectors, CD1d1 S330A -vectors or pcDNA3.1(-) vectors (mock), cells were further treated with LPS and iGb3, and cell extracts were immunoblotted for IκBα and STAT1 phosphorylation. Error bar, SEM. * P

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Protein Binding, Quantitative RT-PCR, Immunoprecipitation, Stable Transfection, Transfection

    18) Product Images from "E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts"

    Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1713-x

    STAT1/MyD88 complexes mediate E2F2 regulation of inflammatory cytokines. a – d STAT1/MyD88 complex is found in RASFs and MEFs. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS; 10 μg/mL). Co-IP ( a , b ) was performed to test the binding. Confocal immunofluorescence ( c , d ) was performed to confirm the result (magnification 10 × 40, MyD88 (green) and STAT1 (red)). e – g Effect of STAT1/MyD88 complexes on the expression of cytokines. qRT-PCR was performed to detect expression of interleukin (IL)-1α ( e ), IL-1β ( f ), and tumor necrosis factor (TNF)-α ( g ) in STAT1/MyD88 knockdown RASFs with or without E2F2 overexpression in the presence of LPS (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. ** P
    Figure Legend Snippet: STAT1/MyD88 complexes mediate E2F2 regulation of inflammatory cytokines. a – d STAT1/MyD88 complex is found in RASFs and MEFs. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS; 10 μg/mL). Co-IP ( a , b ) was performed to test the binding. Confocal immunofluorescence ( c , d ) was performed to confirm the result (magnification 10 × 40, MyD88 (green) and STAT1 (red)). e – g Effect of STAT1/MyD88 complexes on the expression of cytokines. qRT-PCR was performed to detect expression of interleukin (IL)-1α ( e ), IL-1β ( f ), and tumor necrosis factor (TNF)-α ( g ) in STAT1/MyD88 knockdown RASFs with or without E2F2 overexpression in the presence of LPS (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. ** P

    Techniques Used: Cell Culture, Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Over Expression

    STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a – d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR ( a , b ) and Western blot ( c , d ) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP ( f ) and luciferase (Luc) reporter assays ( g ) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs ( h ) and E2f2 −/− MEFs ( i ). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs ( j ) and E2f2 −/− MEFs ( k ). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2 −/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. * P
    Figure Legend Snippet: STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a – d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR ( a , b ) and Western blot ( c , d ) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP ( f ) and luciferase (Luc) reporter assays ( g ) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs ( h ) and E2f2 −/− MEFs ( i ). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs ( j ) and E2f2 −/− MEFs ( k ). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2 −/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. * P

    Techniques Used: Expressing, Infection, Small Interfering RNA, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Translocation Assay, Fluorescence, Microscopy, Staining, Over Expression

    E2F2 participates in RA inflammation through STAT1 and PI3K/AKT/NF-κB pathways. RNA-seq was performed to screen target genes downstream of E2F2 in RASFs. a , b Heat maps indicate the most differentially expressed genes in RASFs with E2F2 knocked-down. Colored bands represent the change in gene expression: red, downregulation; blue, upregulation. c – e In-vitro verification of genes related to inflammation in RA was performed using qRT-PCR. mRNA levels of STAT1 ( c ), AKT1 ( d ), and AKT2 ( e ). f Western blot was performed to test inhibitory effects of siE2F2 on expression of E2F2, STAT1, AKT1, AKT2, p-AKT, and the p65 subunit of NF-κB. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. NC knockdown scramble control, si small interfering
    Figure Legend Snippet: E2F2 participates in RA inflammation through STAT1 and PI3K/AKT/NF-κB pathways. RNA-seq was performed to screen target genes downstream of E2F2 in RASFs. a , b Heat maps indicate the most differentially expressed genes in RASFs with E2F2 knocked-down. Colored bands represent the change in gene expression: red, downregulation; blue, upregulation. c – e In-vitro verification of genes related to inflammation in RA was performed using qRT-PCR. mRNA levels of STAT1 ( c ), AKT1 ( d ), and AKT2 ( e ). f Western blot was performed to test inhibitory effects of siE2F2 on expression of E2F2, STAT1, AKT1, AKT2, p-AKT, and the p65 subunit of NF-κB. All results are presented as the mean ± SEM of three independent experiments performed in triplicate. NC knockdown scramble control, si small interfering

    Techniques Used: RNA Sequencing Assay, Expressing, In Vitro, Quantitative RT-PCR, Western Blot

    19) Product Images from "Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus"

    Article Title: Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092771

    Effect of recombinant IL-21 on expression of CD56, STAT proteins, and IFN-γ in NK-92 cells. NK-92 cells were cultured in the presence or absence of recombinant IL-21 (0.2 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were incubated with or without recombinant IL-21 (0.2 ng/mL) for 6 h and subsequently cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells for 12 h. Cell lysates from NK-92 cells only were assessed for the expressions of STAT1 and STAT5 proteins. β-actin was served as the loading control. IFN-γ production (pg/mL) in culture supernatants was determined by ELISA. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot; ( D ) Relative band intensity of STAT1 and STAT5 proteins compared with the loading control from at least three independent experiments; ( E ) IFN-γ production (pg/mL).
    Figure Legend Snippet: Effect of recombinant IL-21 on expression of CD56, STAT proteins, and IFN-γ in NK-92 cells. NK-92 cells were cultured in the presence or absence of recombinant IL-21 (0.2 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were incubated with or without recombinant IL-21 (0.2 ng/mL) for 6 h and subsequently cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells for 12 h. Cell lysates from NK-92 cells only were assessed for the expressions of STAT1 and STAT5 proteins. β-actin was served as the loading control. IFN-γ production (pg/mL) in culture supernatants was determined by ELISA. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot; ( D ) Relative band intensity of STAT1 and STAT5 proteins compared with the loading control from at least three independent experiments; ( E ) IFN-γ production (pg/mL).

    Techniques Used: Recombinant, Expressing, Cell Culture, Staining, FACS, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

    Effect of anti-IL-10 on expression of CD56, and STAT proteins in NK-92 cells. NK-92 cells were cultured in the presence or absence of anti-IL-10 (0.1 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells in the presence or absence of anti-IL-10 for 6 h. Cell lysates from NK-92 cells only were assessed for the expressions of stat1 and stat5 proteins. β-actin was served as the loading control. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot. ( D ) Relative band intensity of STAT1 and STAT5 compared with the loading control from at least three independent experiments.
    Figure Legend Snippet: Effect of anti-IL-10 on expression of CD56, and STAT proteins in NK-92 cells. NK-92 cells were cultured in the presence or absence of anti-IL-10 (0.1 ng/mL) for 6 h and stained with fluorescent anti-CD56 antibodies. ( A ) Representative FACS plots showing CD56 expression of NK-92; ( B ) % CD56 +dim NK-92. Alternatively, NK-92 cells were cocultured with J6/JFH-1-huh 7.5 cells or naïve huh 7.5 cells in the presence or absence of anti-IL-10 for 6 h. Cell lysates from NK-92 cells only were assessed for the expressions of stat1 and stat5 proteins. β-actin was served as the loading control. ( C ) Expressions of STAT1 and STAT5 proteins by Western blot. ( D ) Relative band intensity of STAT1 and STAT5 compared with the loading control from at least three independent experiments.

    Techniques Used: Expressing, Cell Culture, Staining, FACS, Western Blot

    20) Product Images from "Loss of PARP-1 attenuates diabetic arteriosclerotic calcification via Stat1/Runx2 axis"

    Article Title: Loss of PARP-1 attenuates diabetic arteriosclerotic calcification via Stat1/Runx2 axis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-2215-8

    PARP-1 deficiency attenuates Stat1-mediated macrophage calcification and macrophage polarization. a Immunoblotting was performed to detect the effect of PARP-1 depletion on total and phosphorylated Stat1 levels ( n = 5). b Stat1 depletion attenuates high glucose-promoted macrophage calcification in vitro ( n = 5). Scale bar = 10 µm. c Immunoblotting and flow cytometry were performed to detect the effect of Stat1 depletion on macrophage phenotype switching ( n = 5).
    Figure Legend Snippet: PARP-1 deficiency attenuates Stat1-mediated macrophage calcification and macrophage polarization. a Immunoblotting was performed to detect the effect of PARP-1 depletion on total and phosphorylated Stat1 levels ( n = 5). b Stat1 depletion attenuates high glucose-promoted macrophage calcification in vitro ( n = 5). Scale bar = 10 µm. c Immunoblotting and flow cytometry were performed to detect the effect of Stat1 depletion on macrophage phenotype switching ( n = 5).

    Techniques Used: In Vitro, Flow Cytometry, Cytometry

    Stat1 directly binds to the Runx2 promoter and contributes to PARP-1-mediated arteriosclerotic calcification. a Predicted Stat1 binding site (underlined) within the human Runx2 promoter. Mutants with deletion of the predicted binding site (Runx2-mut1, Runx2-mut2, and Runx2-mut3) are shown. b Luciferase activity assay was performed after transfection with the human Runx2 promoter or Runx2 promoter mutants in 293T cells ( n = 5).* P
    Figure Legend Snippet: Stat1 directly binds to the Runx2 promoter and contributes to PARP-1-mediated arteriosclerotic calcification. a Predicted Stat1 binding site (underlined) within the human Runx2 promoter. Mutants with deletion of the predicted binding site (Runx2-mut1, Runx2-mut2, and Runx2-mut3) are shown. b Luciferase activity assay was performed after transfection with the human Runx2 promoter or Runx2 promoter mutants in 293T cells ( n = 5).* P

    Techniques Used: Binding Assay, Luciferase, Activity Assay, Transfection

    Proposed working schematic of PARP-1 depletion-mediated attenuation of atherosclerotic calcification in diabetes. Diabetes activates Runx2 expression and induces the osteogenic differentiation of both VSMCs and macrophages. Concurrently, diabetes promotes phenotype switching of VSMCs from the contractile phenotype to a dedifferentiated synthetic phenotype, and of macrophages to a proinflammatory M1 phenotype, which in turn aggravates VSMC calcification. PARP-1 acts on Stat1 transcription, which functions as a regulator of Runx2 expression and osteogenic differentiation. PARP-1 depletion reversed the hyperglycemia-induced synthetic phenotype switching of VSMCs and macrophage polarization by targeting Stat1. As a result, PARP-1 depletion suppresses diabetic atherosclerotic calcification.
    Figure Legend Snippet: Proposed working schematic of PARP-1 depletion-mediated attenuation of atherosclerotic calcification in diabetes. Diabetes activates Runx2 expression and induces the osteogenic differentiation of both VSMCs and macrophages. Concurrently, diabetes promotes phenotype switching of VSMCs from the contractile phenotype to a dedifferentiated synthetic phenotype, and of macrophages to a proinflammatory M1 phenotype, which in turn aggravates VSMC calcification. PARP-1 acts on Stat1 transcription, which functions as a regulator of Runx2 expression and osteogenic differentiation. PARP-1 depletion reversed the hyperglycemia-induced synthetic phenotype switching of VSMCs and macrophage polarization by targeting Stat1. As a result, PARP-1 depletion suppresses diabetic atherosclerotic calcification.

    Techniques Used: Expressing

    PARP-1 deficiency attenuates Stat1-mediated VSMCs calcification and synthetic phenotype switching. a Protein–protein interaction networks illustrate the osteogenic genes downstream of osteogenic TFs. b , c Stat1 binding activity following PARP-1 deletion in the aortas of diabetic ApoE −/− mice. n = 3 in each group. Asterisks indicate statistically significant differences (* P
    Figure Legend Snippet: PARP-1 deficiency attenuates Stat1-mediated VSMCs calcification and synthetic phenotype switching. a Protein–protein interaction networks illustrate the osteogenic genes downstream of osteogenic TFs. b , c Stat1 binding activity following PARP-1 deletion in the aortas of diabetic ApoE −/− mice. n = 3 in each group. Asterisks indicate statistically significant differences (* P

    Techniques Used: Binding Assay, Activity Assay, Mouse Assay

    21) Product Images from "IL-21 is required for CD4 memory formation in response to viral infection"

    Article Title: IL-21 is required for CD4 memory formation in response to viral infection

    Journal: JCI Insight

    doi: 10.1172/jci.insight.90652

    IL-21 signaling promotes activation of STAT1 and STAT3.
    Figure Legend Snippet: IL-21 signaling promotes activation of STAT1 and STAT3.

    Techniques Used: Activation Assay

    IL-21–mediated CD4 T cell survival is dependent on STAT1 and STAT3.
    Figure Legend Snippet: IL-21–mediated CD4 T cell survival is dependent on STAT1 and STAT3.

    Techniques Used:

    22) Product Images from "Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection"

    Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

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

    doi: 10.4049/jimmunol.1003009

    IL-21 activates the STAT1 and STAT3 signaling pathway, leading to the enhanced survival of CD8 T cells in vitro. (A) Purified polyclonal CD8 cells were cultured for 1 h in medium alone (Media) or medium supplemented with recombinant murine IL-21 (+ IL21). Cells were stained with anti-CD8 and anti-pSTAT1, anti-pSTAT3, or anti-pSTAT5 intracellularly. (B–C) Purified polyclonal CD8 cells from WT or STAT1 −/− mice were retrovirally infected with empty vector (Control) or STAT3-D vector (STAT3-D) and stimulated with soluble anti-CD3 and anti-CD28 in medium alone (Media) or medium supplemented with IL-21 (+ IL21). (B) 4 days later, cells were stained with anti-CD8 and Annexin V. Percentage of Annexin V + CD8 cells is indicated. Plots are gated on GFP + cells. (C) GFP+ cells were sorted and mRNA was extracted and quantitative PCR was used to measure the expression of Bcl-2 and Bcl-xL. mRNA abundance was normalized to β-actin. Data are representative of two independent experiments. * = p ≤.001.
    Figure Legend Snippet: IL-21 activates the STAT1 and STAT3 signaling pathway, leading to the enhanced survival of CD8 T cells in vitro. (A) Purified polyclonal CD8 cells were cultured for 1 h in medium alone (Media) or medium supplemented with recombinant murine IL-21 (+ IL21). Cells were stained with anti-CD8 and anti-pSTAT1, anti-pSTAT3, or anti-pSTAT5 intracellularly. (B–C) Purified polyclonal CD8 cells from WT or STAT1 −/− mice were retrovirally infected with empty vector (Control) or STAT3-D vector (STAT3-D) and stimulated with soluble anti-CD3 and anti-CD28 in medium alone (Media) or medium supplemented with IL-21 (+ IL21). (B) 4 days later, cells were stained with anti-CD8 and Annexin V. Percentage of Annexin V + CD8 cells is indicated. Plots are gated on GFP + cells. (C) GFP+ cells were sorted and mRNA was extracted and quantitative PCR was used to measure the expression of Bcl-2 and Bcl-xL. mRNA abundance was normalized to β-actin. Data are representative of two independent experiments. * = p ≤.001.

    Techniques Used: In Vitro, Purification, Cell Culture, Recombinant, Staining, Mouse Assay, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing

    IL-21 signaling promotes activation of the STAT1 and STAT3 pathways and upregulation of Bcl-xL in vivo. 1 × 10 4 purified naïve clone 4 CD8 T cells (Thy1.2 + ) from either WT or IL-21R −/− were adoptively transferred into congenic B10.D2 mice (Thy1.1 + ) that were subsequently infected with rVV-HA (5 × 10 5 pfu, i.p.), of left uninfected (Naive). (A) 5 days post-infection, splenocytes were stimulated with PMA and Ionomycin and then stained with anti-CD8, anti-Thy1.2, as well as anti-pSTAT1, anti-pSTAT3, anti-STAT1, anti-STAT3, or an isotype control intracellularly. The percentage of pSTAT1 + and pSTAT3 + among clonotypic CD8 T cells is indicated. (B) 7 days post-infection, splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-Bcl-xL intracellularly. MFI of clonotypic CD8 T cells is indicated. Plots are gated on CD8 + Thy1.2 + cells. Data are representative of three independent experiments.
    Figure Legend Snippet: IL-21 signaling promotes activation of the STAT1 and STAT3 pathways and upregulation of Bcl-xL in vivo. 1 × 10 4 purified naïve clone 4 CD8 T cells (Thy1.2 + ) from either WT or IL-21R −/− were adoptively transferred into congenic B10.D2 mice (Thy1.1 + ) that were subsequently infected with rVV-HA (5 × 10 5 pfu, i.p.), of left uninfected (Naive). (A) 5 days post-infection, splenocytes were stimulated with PMA and Ionomycin and then stained with anti-CD8, anti-Thy1.2, as well as anti-pSTAT1, anti-pSTAT3, anti-STAT1, anti-STAT3, or an isotype control intracellularly. The percentage of pSTAT1 + and pSTAT3 + among clonotypic CD8 T cells is indicated. (B) 7 days post-infection, splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-Bcl-xL intracellularly. MFI of clonotypic CD8 T cells is indicated. Plots are gated on CD8 + Thy1.2 + cells. Data are representative of three independent experiments.

    Techniques Used: Activation Assay, In Vivo, Purification, Mouse Assay, Infection, Staining

    23) Product Images from "TACI-Deficient Macrophages Protect Mice Against Metaflammation and Obesity-Induced Dysregulation of Glucose Homeostasis"

    Article Title: TACI-Deficient Macrophages Protect Mice Against Metaflammation and Obesity-Induced Dysregulation of Glucose Homeostasis

    Journal: Diabetes

    doi: 10.2337/db17-1089

    Impaired BAFF- and APRIL-induced ERK/STAT1 signaling is responsible for the M2 polarization of TACI-deficient Mϕs. A : AT Mϕs from TACI KO or WT HFD mice were stimulated with APRIL plus BAFF for 24 h. Total and phosphorylated ERK1/2 and STAT1 were detected by Western blot analysis. One out of three experiments with similar results is shown. B : Peritoneal Mϕs from wild-type (WT) mice were stimulated with APRIL plus BAFF for 24 h after transfection with control or STAT1 siRNA. Total and phosphorylated forms of STAT1 and ERK1/2 were detected by Western blot analysis. One out of three experiments with similar results is shown. C : The expression of M1-associated genes in these cells was analyzed by real-time PCR. Data presented are the mean ± SD of three experiments. ns, not significant. * P
    Figure Legend Snippet: Impaired BAFF- and APRIL-induced ERK/STAT1 signaling is responsible for the M2 polarization of TACI-deficient Mϕs. A : AT Mϕs from TACI KO or WT HFD mice were stimulated with APRIL plus BAFF for 24 h. Total and phosphorylated ERK1/2 and STAT1 were detected by Western blot analysis. One out of three experiments with similar results is shown. B : Peritoneal Mϕs from wild-type (WT) mice were stimulated with APRIL plus BAFF for 24 h after transfection with control or STAT1 siRNA. Total and phosphorylated forms of STAT1 and ERK1/2 were detected by Western blot analysis. One out of three experiments with similar results is shown. C : The expression of M1-associated genes in these cells was analyzed by real-time PCR. Data presented are the mean ± SD of three experiments. ns, not significant. * P

    Techniques Used: Mouse Assay, Western Blot, Transfection, Expressing, Real-time Polymerase Chain Reaction

    24) Product Images from "The C-Terminal Transactivation Domain of STAT1 Has a Gene-Specific Role in Transactivation and Cofactor Recruitment"

    Article Title: The C-Terminal Transactivation Domain of STAT1 Has a Gene-Specific Role in Transactivation and Cofactor Recruitment

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02879

    STAT1 and IRF1 binding to the Irf7 and Irf8 promoters and H3ac and H4ac and H3K4me3 around the Irf1, Irf8 , and Irf7 TSS before and after IFNγ treatment. (A–C) Schematic representation of the murine Irf7 (A) , Irf1 (B) , and Irf8 (C) promoter regions. GAS and ISRE sites, the TSS and the position of the primers used for the ChIP analyses are depicted. (D–O) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). STAT1 and STAT2 binding at the Irf7 ISRE (D,E) and STAT1 binding at the Irf8 (F) GAS element was analyzed by ChIP. H3 pan-acetylation (H3ac), H4 pan-acetylation (H4ac), and H3 lysine 4 trimethylation (H3K4me3) around the Irf1 (G–I) , the Irf8 (J–L) , and the Irf7 (M–O) TSS was determined by ChIP. Data were normalized to the input control (D–F) or the total levels of H3 (G–O) . Mean values ± SE from three to four independent experiments are shown; ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.
    Figure Legend Snippet: STAT1 and IRF1 binding to the Irf7 and Irf8 promoters and H3ac and H4ac and H3K4me3 around the Irf1, Irf8 , and Irf7 TSS before and after IFNγ treatment. (A–C) Schematic representation of the murine Irf7 (A) , Irf1 (B) , and Irf8 (C) promoter regions. GAS and ISRE sites, the TSS and the position of the primers used for the ChIP analyses are depicted. (D–O) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). STAT1 and STAT2 binding at the Irf7 ISRE (D,E) and STAT1 binding at the Irf8 (F) GAS element was analyzed by ChIP. H3 pan-acetylation (H3ac), H4 pan-acetylation (H4ac), and H3 lysine 4 trimethylation (H3K4me3) around the Irf1 (G–I) , the Irf8 (J–L) , and the Irf7 (M–O) TSS was determined by ChIP. Data were normalized to the input control (D–F) or the total levels of H3 (G–O) . Mean values ± SE from three to four independent experiments are shown; ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Mouse Assay

    Recruitment and phosphorylation of Pol II at the Irf8 and Irf7 genes and occupancy of Mediator components at the Irf8 promoter. (A–K) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf8 (A–C) and the Irf7 promoters (H–J) around the TSS and of S2pPol II within the respective gene bodies (D,K) was determined by ChIP. The promoter occupancy of MED26 (E) , MED1 (F) , and MED24 (G) at the Irf8 GAS. Data were normalized to the input control. Mean values ± SE from two (K) , three (A–I) or four (J) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 .
    Figure Legend Snippet: Recruitment and phosphorylation of Pol II at the Irf8 and Irf7 genes and occupancy of Mediator components at the Irf8 promoter. (A–K) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf8 (A–C) and the Irf7 promoters (H–J) around the TSS and of S2pPol II within the respective gene bodies (D,K) was determined by ChIP. The promoter occupancy of MED26 (E) , MED1 (F) , and MED24 (G) at the Irf8 GAS. Data were normalized to the input control. Mean values ± SE from two (K) , three (A–I) or four (J) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 .

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation

    STAT1 and IRF1 binding to the CIIta promoter IV (pIV) and the Gbp2 promoters and IFNγ-induced histone modifications. (A,B) Schematic representation of the murine CIIta and Gbp2 promoter regions. STAT1 and IRF1 binding sites, the TSS, and the position of the primers used for the ChIP analyses are depicted. (C–M) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). STAT1 and IRF1 binding to the CIIta (C, D) and the Gbp2 (E–G) promoter binding sites was analyzed by ChIP. H3 pan-acetylation (H3ac), H4 pan-acetylation (H4ac), and H3 lysine 4 trimethylation (H3K4me3) around the CIIta (H–J) and the Gbp2 (K–M) TSS was determined by ChIP. Data were normalized to the input control (C–G) and the total levels of H3 (H–M) . Mean values ± SE from three (C–G , H,J,K,M) or four (I,L) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.
    Figure Legend Snippet: STAT1 and IRF1 binding to the CIIta promoter IV (pIV) and the Gbp2 promoters and IFNγ-induced histone modifications. (A,B) Schematic representation of the murine CIIta and Gbp2 promoter regions. STAT1 and IRF1 binding sites, the TSS, and the position of the primers used for the ChIP analyses are depicted. (C–M) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). STAT1 and IRF1 binding to the CIIta (C, D) and the Gbp2 (E–G) promoter binding sites was analyzed by ChIP. H3 pan-acetylation (H3ac), H4 pan-acetylation (H4ac), and H3 lysine 4 trimethylation (H3K4me3) around the CIIta (H–J) and the Gbp2 (K–M) TSS was determined by ChIP. Data were normalized to the input control (C–G) and the total levels of H3 (H–M) . Mean values ± SE from three (C–G , H,J,K,M) or four (I,L) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Mouse Assay

    Promoter occupancy of Pol II, S5 phosphorylated Pol II (S5pPol II), and S2 phosphorylated Pol II (S2pPol II) around the CIIta and Gbp2 TSS and of S2pPol II at the Gbp2 gene body. BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). The association of Pol II, S5pPol II, and S2pPol II with the TSS of (A–C) CIIta pIV and (D–F) Gbp2 TSS, and (G) association of S2pPol II with the Gbp2 gene body was determined by ChIP. Data were normalized to the input control. Mean values ± SE from three (D,G) , four (A,B) or five (C,E,F) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.
    Figure Legend Snippet: Promoter occupancy of Pol II, S5 phosphorylated Pol II (S5pPol II), and S2 phosphorylated Pol II (S2pPol II) around the CIIta and Gbp2 TSS and of S2pPol II at the Gbp2 gene body. BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). The association of Pol II, S5pPol II, and S2pPol II with the TSS of (A–C) CIIta pIV and (D–F) Gbp2 TSS, and (G) association of S2pPol II with the Gbp2 gene body was determined by ChIP. Data were normalized to the input control. Mean values ± SE from three (D,G) , four (A,B) or five (C,E,F) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation

    IFNγ induced expression of Irf1 and Irf7 in Stat1 β/β cells in the absence of STAT2 or IRF9. BMDMs derived from Stat1 β/β , Stat1 β/β Stat2 −/− and Stat1 β/β Irf9 −/− mice were stimulated with IFNγ for the times indicated or left untreated (0 h, -). (A) Protein was isolated and Tyr701-phosphorylated STAT1 (pSTAT1) and STAT1 protein levels determined by Western blotting. ERK p42 was used as loading control. One representative out of three independent experiments is shown. Original uncropped blots are shown in Supplementary Figure 2 . (B,C) Total RNA was isolated and Irf1 (B) and Irf7 (C) mRNA expression was determined by RT-qPCR. Data were normalized to Ube2d2 . Mean values ± SE from three (C) or four (B) independent experiments are shown. * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.
    Figure Legend Snippet: IFNγ induced expression of Irf1 and Irf7 in Stat1 β/β cells in the absence of STAT2 or IRF9. BMDMs derived from Stat1 β/β , Stat1 β/β Stat2 −/− and Stat1 β/β Irf9 −/− mice were stimulated with IFNγ for the times indicated or left untreated (0 h, -). (A) Protein was isolated and Tyr701-phosphorylated STAT1 (pSTAT1) and STAT1 protein levels determined by Western blotting. ERK p42 was used as loading control. One representative out of three independent experiments is shown. Original uncropped blots are shown in Supplementary Figure 2 . (B,C) Total RNA was isolated and Irf1 (B) and Irf7 (C) mRNA expression was determined by RT-qPCR. Data were normalized to Ube2d2 . Mean values ± SE from three (C) or four (B) independent experiments are shown. * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.

    Techniques Used: Expressing, Derivative Assay, Mouse Assay, Isolation, Western Blot, Quantitative RT-PCR

    Recruitment and phosphorylation of Pol II at Irf1 gene and occupancy of TFIIH, p-TEFb, and Mediator components at the Irf1 TSS. (A) Schematic representation of Pol II, GTFs, and the Mediator complex at a GAS-driven gene promoter. Components of the Mediator, TFIIH, and p-TEFb complexes analyzed by ChIP are indicated. (B–L) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf1 promoter around the TSS (B–D) and of S2pPol II within the Irf1 gene body (E) . Association of ERCC3 (F) and CDK9 (G) at the Irf1 TSS and of MED18 (H) , MED4 (I) , MED24 (J) , MED26 (K) , and MED1 (L) at the Irf1 GAS. Data were normalized to the input control. Mean values ± SE from two (H) or three (all others) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 .
    Figure Legend Snippet: Recruitment and phosphorylation of Pol II at Irf1 gene and occupancy of TFIIH, p-TEFb, and Mediator components at the Irf1 TSS. (A) Schematic representation of Pol II, GTFs, and the Mediator complex at a GAS-driven gene promoter. Components of the Mediator, TFIIH, and p-TEFb complexes analyzed by ChIP are indicated. (B–L) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf1 promoter around the TSS (B–D) and of S2pPol II within the Irf1 gene body (E) . Association of ERCC3 (F) and CDK9 (G) at the Irf1 TSS and of MED18 (H) , MED4 (I) , MED24 (J) , MED26 (K) , and MED1 (L) at the Irf1 GAS. Data were normalized to the input control. Mean values ± SE from two (H) or three (all others) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 .

    Techniques Used: Chromatin Immunoprecipitation, Mouse Assay

    Transcriptional activity of STAT1β at the Irf1, Irf8, CIIta, Gbp2 , and Irf7 genes. BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h, -). Total RNA (A–D, G) or protein extracts (E) were isolated. Irf1 (A) , Irf8 (B) , CIIta (C) , Gbp2 (D) , and Irf7 (G) pre-mRNA expression was determined by RT-qPCR. Data were normalized to the housekeeping gene Ube2d2 . Mean values ± SE from three independent experiments are shown. (E) IRF1 protein levels were determined by Western blotting. ERK p42 was used as loading control. Data are as representative of two independent experiments. Original uncropped blots are shown in Supplementary Figure 1 . (F) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for 24 h. MHC class II surface levels were determined by flow cytometry. Median fluorescence intensities (MdFI) ±standard error (SE) from two independent experiments are shown. (A–D, F–G) * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.
    Figure Legend Snippet: Transcriptional activity of STAT1β at the Irf1, Irf8, CIIta, Gbp2 , and Irf7 genes. BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h, -). Total RNA (A–D, G) or protein extracts (E) were isolated. Irf1 (A) , Irf8 (B) , CIIta (C) , Gbp2 (D) , and Irf7 (G) pre-mRNA expression was determined by RT-qPCR. Data were normalized to the housekeeping gene Ube2d2 . Mean values ± SE from three independent experiments are shown. (E) IRF1 protein levels were determined by Western blotting. ERK p42 was used as loading control. Data are as representative of two independent experiments. Original uncropped blots are shown in Supplementary Figure 1 . (F) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for 24 h. MHC class II surface levels were determined by flow cytometry. Median fluorescence intensities (MdFI) ±standard error (SE) from two independent experiments are shown. (A–D, F–G) * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 . Significances are only indicated for the comparisons between genotypes.

    Techniques Used: Activity Assay, Mouse Assay, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Cytometry, Fluorescence

    25) Product Images from "STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling"

    Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.3378

    Inhibiting negative feedback regulation of USP18 by targeting its interaction with STAT2 (a) 293T cells were co-transfected with plasmids encoding STAT1-FLAG, FLAG-USP18 and either STAT2 CC/DB-Myc or the mutant STAT2 CC/DB L227A R409A K415A-Myc (3A). Following treatment with IFNα (1000 U/ml) for 15 minutes as indicated cell lysates were collected and immunoblotted with indicated antibodies. The ratio of p-STAT1/total STAT1 was quantified by LI-COR Odyssey system. (b) Cells indicated in Fig. 7a were treated with IFNα (1000 U/ml) for 12 hours and then expression of GBP1 was analyzed by RT q-PCR. Data represents mean ± S.D. for two independent experiments. (c) THP-1 cells transduced with either MIP control (−) or MIP-STAT2 CC/DB 3A (3A) were treated with IFNα (1000 U/ml) for 48 hours and then Annexin V positive cells were analyzed by flow cytometry. Data represents mean ± S.E.M. for three independently generated stable cell lines. ** P
    Figure Legend Snippet: Inhibiting negative feedback regulation of USP18 by targeting its interaction with STAT2 (a) 293T cells were co-transfected with plasmids encoding STAT1-FLAG, FLAG-USP18 and either STAT2 CC/DB-Myc or the mutant STAT2 CC/DB L227A R409A K415A-Myc (3A). Following treatment with IFNα (1000 U/ml) for 15 minutes as indicated cell lysates were collected and immunoblotted with indicated antibodies. The ratio of p-STAT1/total STAT1 was quantified by LI-COR Odyssey system. (b) Cells indicated in Fig. 7a were treated with IFNα (1000 U/ml) for 12 hours and then expression of GBP1 was analyzed by RT q-PCR. Data represents mean ± S.D. for two independent experiments. (c) THP-1 cells transduced with either MIP control (−) or MIP-STAT2 CC/DB 3A (3A) were treated with IFNα (1000 U/ml) for 48 hours and then Annexin V positive cells were analyzed by flow cytometry. Data represents mean ± S.E.M. for three independently generated stable cell lines. ** P

    Techniques Used: Transfection, Mutagenesis, Expressing, Polymerase Chain Reaction, Transduction, Flow Cytometry, Cytometry, Generated, Stable Transfection

    STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA lentivirus. After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.
    Figure Legend Snippet: STAT2 is required for USP18-mediated inhibition of Type I IFN signaling (a–f) MIP or MIP-USP18 expressing 2fTGH, U1A, U2A, U4A, U5A, and U6A cells were treated with IFNα (1000 U/ml) for 15 minutes. The cell lysates were immunoblotted with the indicated antibodies. (g) IB analysis of STAT1 phosphorylation in 2fTGH and U6A cells in the presence or absence of (1000 U/ml) IFNα. (h–i) Stat2 −/− MEFs were infected with MIP control (−) or MIP-Usp18 (+) retroviruses, either in the presence or absence of rescue with C-terminally FLAG-tagged Stat2 cDNA. Where indicated cells were treated with either mouse IFNβ (500 U/ml) for 15 minutes (h) or 30 minutes (i), before cell lysates were collected and analyzed by Western blotting with indicated antibodies. (j) Validation of Stat2 knockdown. Ba/F3 cells were infected with control or Stat2-targeting shRNA lentivirus. After 5 days puromycin selection, Stat2 expression was examined by Western blotting. (k) Usp18 −/− bone marrow cells were infected with pCX4-bsr control (−) or pCX4-bsr-Usp18 (+), either in the presence or absence of control (shCtrl) or Stat2-knockdown (shStat2–3) shRNA expression. Two days following double drug selection (puromycin and blasticidin), cells were either left untreated (−) or treated (+) with mouse IFNβ (500 U/ml) for 30 minutes. Cell lysates were collected and analyzed by Western blotting with indicated antibodies.

    Techniques Used: Inhibition, Expressing, Infection, Western Blot, shRNA, Selection

    26) Product Images from "Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity"

    Article Title: Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17661

    Cisplatin prevents STAT phosphorylation by binding to STAT proteins and blocking the SH2 domain ( A ) DU-145 cells were cultured with and without co-administration of cisplatin (10 μg/ml) for 24 hours and the cell surface expression of the IL-4, IFNγ and IL-6 receptor was determined by flow cytometry. Shown is one representative experiment out of two independent experiments. ( B ) DU-145 cells were cultured with and without co-administration of cisplatin (10 μg/ml) for 24 hours and the expression of SOCS1, SOCS2, SOCS3, PIAS1 and PIAS3 was determined by flow cytometry. Shown is one representative experiment performed in triplicate (+SEM) out of 2 independent experiments. ( C ) DU-145 cells cultured with IL-4 and with and without simultaneous co-administration of cisplatin (10 μg/ml) cisplatin in the presence of Sodium Stibogluconate (50 μg/ml), NSC87887 (50 μM) or Suramin (100 μg/ml). Phosphorylation of STAT6, 3 and 1 was analyzed by western blot. Shown is one representative experiment out of two independent experiments. ( D ) BLM cells were treated with cisplatin (20 μg/ml) for the indicated times after which the cells were stimulated with IL-6 and IFNγ (STAT1 and STAT3) or IL-4 (STAT6) for 30 minutes. STAT phosphorylation was measured by flow cytometry. Shown is the mean ± SEM of the mean fluorescence of one representative experiment out of at least 2 independent flow cytometry experiments performed in triplicate. ( E ) Binding of pTyr peptide to STAT SH2 domain visualized by normalized energy transfer between donor bead-coupled STATs and acceptor bead-coupled pTyr peptide (alpha screen). STATs were preincubated with increasing concentration of cisplatin (1, 3, 10 or 30 μM). Shown is the mean ± SEM of one representative experiment performed in triplicate out of 3 independent experiments.
    Figure Legend Snippet: Cisplatin prevents STAT phosphorylation by binding to STAT proteins and blocking the SH2 domain ( A ) DU-145 cells were cultured with and without co-administration of cisplatin (10 μg/ml) for 24 hours and the cell surface expression of the IL-4, IFNγ and IL-6 receptor was determined by flow cytometry. Shown is one representative experiment out of two independent experiments. ( B ) DU-145 cells were cultured with and without co-administration of cisplatin (10 μg/ml) for 24 hours and the expression of SOCS1, SOCS2, SOCS3, PIAS1 and PIAS3 was determined by flow cytometry. Shown is one representative experiment performed in triplicate (+SEM) out of 2 independent experiments. ( C ) DU-145 cells cultured with IL-4 and with and without simultaneous co-administration of cisplatin (10 μg/ml) cisplatin in the presence of Sodium Stibogluconate (50 μg/ml), NSC87887 (50 μM) or Suramin (100 μg/ml). Phosphorylation of STAT6, 3 and 1 was analyzed by western blot. Shown is one representative experiment out of two independent experiments. ( D ) BLM cells were treated with cisplatin (20 μg/ml) for the indicated times after which the cells were stimulated with IL-6 and IFNγ (STAT1 and STAT3) or IL-4 (STAT6) for 30 minutes. STAT phosphorylation was measured by flow cytometry. Shown is the mean ± SEM of the mean fluorescence of one representative experiment out of at least 2 independent flow cytometry experiments performed in triplicate. ( E ) Binding of pTyr peptide to STAT SH2 domain visualized by normalized energy transfer between donor bead-coupled STATs and acceptor bead-coupled pTyr peptide (alpha screen). STATs were preincubated with increasing concentration of cisplatin (1, 3, 10 or 30 μM). Shown is the mean ± SEM of one representative experiment performed in triplicate out of 3 independent experiments.

    Techniques Used: Binding Assay, Blocking Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Western Blot, Fluorescence, Concentration Assay

    Platinum drugs specifically inhibit phosphorylation of STATs in multiple cancers ( A ) DU-145 cells were treated with the following cytokines: IL-6/IFNγ (STAT1 and STAT3), IFNα (STAT2), IL-4/IL-13/IFNγ (STAT5) or IL-4 (STAT6), to induce STAT protein phosphorylation, with and without simultaneous co-administration of cisplatin (10 μg/ml) for 18 hours. STAT protein expression and phosphorylation was analyzed by western blot. Actin is shown as a loading control. Shown is one representative experiment out of at least four independent experiments. ( B ) DU-145 cells were treated with IL-4 to induce STAT6 phosphorylation with and without co-administration of cisplatin (10 μg/ml), oxaliplatin (14 or 25 μg/ml), or carboplatin (80 or 120 μg/ml) for 18 hours. Shown is one representative experiment performed in triplicate (+SEM) out of at least three independent experiments. ( C ) BLM (melanoma), DU-145 (prostate cancer), KATO-3 (gastric cancer), HT-29 (colon cancer) and HeLa (cervical cancer of the uterus) cells were treated with IL-4 or IL-6/IFNγ to induce STAT6 or STAT1 and 3 protein phosphorylation, respectively, with and without simultaneous co-administration of cisplatin (10 μg/ml; BLM cells 20 μg/ml) for 18 hours. STAT protein expression and phosphorylation was analyzed by western blot. Shown is one representative experiment out of at least 3 independent experiments. ( D ) DU-145 cells were treated with cytokines: IL-6 and IFNγ (STAT1 and STAT3) with and without co-administration of cisplatin (10 μg/ml) for 18 hours and STAT protein localization was visualized by confocal microscopy. Shown is one representative experiment out 3 independent experiments. ( E ) DU-145 cells were treated with IL-4 to induce STAT6 protein phosphorylation with and without co-administration of cisplatin (10 μg/ml), methotrexate (250 μg/ml), gemcitabine (20 μg/ml), cyclophosphamide (140 μg/ml) or vincristine (0.4 μg/ml) for 18 hours. STAT6 protein expression and phosphorylation were analyzed by western blot. Shown is one representative experiment out of 2 independent experiments. ( F ) DU-145 cells were pretreated or not with cisplatin for 12 hours and pulsed with a cytokine mix (IL-1β, TNF-α, IFNγ) for 30 minutes. ERK phosphorylation was analyzed by flow cytometry. Shown is one representative FACS plot and one representative experiment performed in triplicate (+SEM) out of 2 independent experiments. ( G ) DU-145 cells were treated with IL-4 to induce STAT6 protein phosphorylation with and without co-administration of cisplatin (10 μg/ml) for 18 hours. Akt protein expression and phosphorylation was analyzed by western blot. Shown is one representative experiment out of 2 independent experiments.
    Figure Legend Snippet: Platinum drugs specifically inhibit phosphorylation of STATs in multiple cancers ( A ) DU-145 cells were treated with the following cytokines: IL-6/IFNγ (STAT1 and STAT3), IFNα (STAT2), IL-4/IL-13/IFNγ (STAT5) or IL-4 (STAT6), to induce STAT protein phosphorylation, with and without simultaneous co-administration of cisplatin (10 μg/ml) for 18 hours. STAT protein expression and phosphorylation was analyzed by western blot. Actin is shown as a loading control. Shown is one representative experiment out of at least four independent experiments. ( B ) DU-145 cells were treated with IL-4 to induce STAT6 phosphorylation with and without co-administration of cisplatin (10 μg/ml), oxaliplatin (14 or 25 μg/ml), or carboplatin (80 or 120 μg/ml) for 18 hours. Shown is one representative experiment performed in triplicate (+SEM) out of at least three independent experiments. ( C ) BLM (melanoma), DU-145 (prostate cancer), KATO-3 (gastric cancer), HT-29 (colon cancer) and HeLa (cervical cancer of the uterus) cells were treated with IL-4 or IL-6/IFNγ to induce STAT6 or STAT1 and 3 protein phosphorylation, respectively, with and without simultaneous co-administration of cisplatin (10 μg/ml; BLM cells 20 μg/ml) for 18 hours. STAT protein expression and phosphorylation was analyzed by western blot. Shown is one representative experiment out of at least 3 independent experiments. ( D ) DU-145 cells were treated with cytokines: IL-6 and IFNγ (STAT1 and STAT3) with and without co-administration of cisplatin (10 μg/ml) for 18 hours and STAT protein localization was visualized by confocal microscopy. Shown is one representative experiment out 3 independent experiments. ( E ) DU-145 cells were treated with IL-4 to induce STAT6 protein phosphorylation with and without co-administration of cisplatin (10 μg/ml), methotrexate (250 μg/ml), gemcitabine (20 μg/ml), cyclophosphamide (140 μg/ml) or vincristine (0.4 μg/ml) for 18 hours. STAT6 protein expression and phosphorylation were analyzed by western blot. Shown is one representative experiment out of 2 independent experiments. ( F ) DU-145 cells were pretreated or not with cisplatin for 12 hours and pulsed with a cytokine mix (IL-1β, TNF-α, IFNγ) for 30 minutes. ERK phosphorylation was analyzed by flow cytometry. Shown is one representative FACS plot and one representative experiment performed in triplicate (+SEM) out of 2 independent experiments. ( G ) DU-145 cells were treated with IL-4 to induce STAT6 protein phosphorylation with and without co-administration of cisplatin (10 μg/ml) for 18 hours. Akt protein expression and phosphorylation was analyzed by western blot. Shown is one representative experiment out of 2 independent experiments.

    Techniques Used: Expressing, Western Blot, Confocal Microscopy, Flow Cytometry, Cytometry, FACS

    27) Product Images from "Venous malformation-causative TIE2 mutations mediate an AKT-dependent decrease in PDGFB"

    Article Title: Venous malformation-causative TIE2 mutations mediate an AKT-dependent decrease in PDGFB

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddt198

    Mutant TIE2 causes ligand-independent activation of AKT and STAT1. Western blots on lysates from confluent HUVECs: non-transfected (NT); or retrovirally transfected to overexpress WT-TIE2 (WT), R849W or L914F. Blots probed for pan phospho-tyrosine (phospho-Tyr)
    Figure Legend Snippet: Mutant TIE2 causes ligand-independent activation of AKT and STAT1. Western blots on lysates from confluent HUVECs: non-transfected (NT); or retrovirally transfected to overexpress WT-TIE2 (WT), R849W or L914F. Blots probed for pan phospho-tyrosine (phospho-Tyr)

    Techniques Used: Mutagenesis, Activation Assay, Western Blot, Transfection

    28) Product Images from "Opposing roles of STAT1 and STAT3 in T cell-mediated hepatitis: regulation by SOCS"

    Article Title: Opposing roles of STAT1 and STAT3 in T cell-mediated hepatitis: regulation by SOCS

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI15841

    STAT3 and SOCS3 activation are attenuated, but liver injury, STAT1, IRF-1, and SOCS1 activation are enhanced in Con A–induced hepatitis in IL-6 –/– mice. ( a ) IL-6 +/+ mice and IL-6 –/– mice were injected with 10 μg/g of Con A. At various time points, liver protein extracts and RNA were analyzed by Western blot or RT-PCR (indicated by asterisks), respectively, using Ab’s and primers as indicated (left panel), and quantified by PhosphorImager analysis (right panel). The values are shown as means ± SEM from four independent experiments. * P
    Figure Legend Snippet: STAT3 and SOCS3 activation are attenuated, but liver injury, STAT1, IRF-1, and SOCS1 activation are enhanced in Con A–induced hepatitis in IL-6 –/– mice. ( a ) IL-6 +/+ mice and IL-6 –/– mice were injected with 10 μg/g of Con A. At various time points, liver protein extracts and RNA were analyzed by Western blot or RT-PCR (indicated by asterisks), respectively, using Ab’s and primers as indicated (left panel), and quantified by PhosphorImager analysis (right panel). The values are shown as means ± SEM from four independent experiments. * P

    Techniques Used: Activation Assay, Mouse Assay, Injection, Western Blot, Reverse Transcription Polymerase Chain Reaction

    A model illustrating opposing roles of STAT1 and STAT3 in Con A–induced hepatitis. Con A activates multiple immune cells, including NK T cells and CD4 + T cells, and induces the release of a variety of cytokines. IFN-γ induces activation of JAK1 and JAK2 and consequent activation of STAT1. IFN-γ/STAT1 play an essential role in CD4 + and NK T cell activation, which, in turn, directly or indirectly induce liver injury. IFN-γ/STAT1 also induce expression of proapoptotic IRF-1 protein, which mediates liver apoptosis and injury. IL-6 and IFN-γ activate JAK1 and JAK2 and consequently induce STAT3 activation, followed by induction of Bcl-X L and other antiapoptotic factors, which protect against hepatic necrosis and apoptosis. IL-6 activation of STAT3 also downregulates IFN-γ and IFN-γ signaling and consequently suppresses IFN-γ/STAT1-induced liver injury. Activated STAT1 and STAT3 inhibit one another, at least in hepatocytes, by the induction of SOCS.
    Figure Legend Snippet: A model illustrating opposing roles of STAT1 and STAT3 in Con A–induced hepatitis. Con A activates multiple immune cells, including NK T cells and CD4 + T cells, and induces the release of a variety of cytokines. IFN-γ induces activation of JAK1 and JAK2 and consequent activation of STAT1. IFN-γ/STAT1 play an essential role in CD4 + and NK T cell activation, which, in turn, directly or indirectly induce liver injury. IFN-γ/STAT1 also induce expression of proapoptotic IRF-1 protein, which mediates liver apoptosis and injury. IL-6 and IFN-γ activate JAK1 and JAK2 and consequently induce STAT3 activation, followed by induction of Bcl-X L and other antiapoptotic factors, which protect against hepatic necrosis and apoptosis. IL-6 activation of STAT3 also downregulates IFN-γ and IFN-γ signaling and consequently suppresses IFN-γ/STAT1-induced liver injury. Activated STAT1 and STAT3 inhibit one another, at least in hepatocytes, by the induction of SOCS.

    Techniques Used: Activation Assay, Expressing

    Liver injury and STAT1 and SOCS1 activation are attenuated, but STAT3 and SOCS3 activation are enhanced and prolonged in Con A–induced hepatitis in STAT1 –/– mice. ( a ) Mice were injected with 22 μg/g of Con A. At various time points, serum ALT levels were measured. Values are shown as means ± SEM from four mice at each time point. ( b ) Photomicrographs of representative mouse livers from 9-hour Con A–treated wild-type and STAT1 –/– mice with H E staining are shown (original magnification ×200 and ×400). White arrows indicate massive necrosis observed in the liver. ( c ) Wild-type control and STAT1 –/– mice were injected with Con A (22 μg/g). At various time points after injection, serum was collected, and circulating IFN-γ levels were measured by ELISA. Values are shown as means ± SEM from three mice at each time point. ( d ) Total liver protein extracts and RNA from Con A–treated STAT1 +/+ and STAT1 –/– mice were analyzed by Western blotting and RT-PCR (indicated by asterisks), respectively, using Ab’s and primers as indicated. Induction of pSTAT1, pSTAT3, IRF-1, Bcl-X L , SOCS1, and SOCS3 was quantified by PhosphorImager analysis (left panel), as described in Methods. The values are shown as means ± SEM from four independent experiments at each time point. * P
    Figure Legend Snippet: Liver injury and STAT1 and SOCS1 activation are attenuated, but STAT3 and SOCS3 activation are enhanced and prolonged in Con A–induced hepatitis in STAT1 –/– mice. ( a ) Mice were injected with 22 μg/g of Con A. At various time points, serum ALT levels were measured. Values are shown as means ± SEM from four mice at each time point. ( b ) Photomicrographs of representative mouse livers from 9-hour Con A–treated wild-type and STAT1 –/– mice with H E staining are shown (original magnification ×200 and ×400). White arrows indicate massive necrosis observed in the liver. ( c ) Wild-type control and STAT1 –/– mice were injected with Con A (22 μg/g). At various time points after injection, serum was collected, and circulating IFN-γ levels were measured by ELISA. Values are shown as means ± SEM from three mice at each time point. ( d ) Total liver protein extracts and RNA from Con A–treated STAT1 +/+ and STAT1 –/– mice were analyzed by Western blotting and RT-PCR (indicated by asterisks), respectively, using Ab’s and primers as indicated. Induction of pSTAT1, pSTAT3, IRF-1, Bcl-X L , SOCS1, and SOCS3 was quantified by PhosphorImager analysis (left panel), as described in Methods. The values are shown as means ± SEM from four independent experiments at each time point. * P

    Techniques Used: Activation Assay, Mouse Assay, Injection, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    IFN-γ and IL-6 induce prolonged activation of STAT3 in STAT1 –/– mouse hepatocytes. Primary cultured mouse hepatocytes were treated with IFN-γ (10 ng/ml) or IL-6 (20 ng/ml). At various time points, as indicated, cell protein extracts and RNA were prepared and analyzed by Western blotting and RT-PCR (indicated by asterisks), respectively, using Ab’s or primers as indicated. Similar data were obtained from five independent experiments.
    Figure Legend Snippet: IFN-γ and IL-6 induce prolonged activation of STAT3 in STAT1 –/– mouse hepatocytes. Primary cultured mouse hepatocytes were treated with IFN-γ (10 ng/ml) or IL-6 (20 ng/ml). At various time points, as indicated, cell protein extracts and RNA were prepared and analyzed by Western blotting and RT-PCR (indicated by asterisks), respectively, using Ab’s or primers as indicated. Similar data were obtained from five independent experiments.

    Techniques Used: Activation Assay, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Liver injury, STAT1 and SOCS1 activation are attenuated, whereas STAT3 activation is slightly enhanced in Con A–induced hepatitis in IFN- γ –/– mice. ( a ) IFN-γ +/+ mice and IFN-γ –/– mice were injected with 15 μg/g of Con A. At various time points, total liver protein extracts and RNA were prepared and analyzed by Western blotting and RT-PCR (indicated by asterisks), respectively, using Ab’s and primers as indicated. Induction of pSTAT1, pSTAT3, IRF-1, Bcl-X L , SOCS1, and SOCS3 was quantified by PhosphorImager analysis (left panel), as described in Methods. The values are shown as means ± SEM from three independent experiments at each time point. * P
    Figure Legend Snippet: Liver injury, STAT1 and SOCS1 activation are attenuated, whereas STAT3 activation is slightly enhanced in Con A–induced hepatitis in IFN- γ –/– mice. ( a ) IFN-γ +/+ mice and IFN-γ –/– mice were injected with 15 μg/g of Con A. At various time points, total liver protein extracts and RNA were prepared and analyzed by Western blotting and RT-PCR (indicated by asterisks), respectively, using Ab’s and primers as indicated. Induction of pSTAT1, pSTAT3, IRF-1, Bcl-X L , SOCS1, and SOCS3 was quantified by PhosphorImager analysis (left panel), as described in Methods. The values are shown as means ± SEM from three independent experiments at each time point. * P

    Techniques Used: Activation Assay, Mouse Assay, Injection, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Con A injection–mediated activation of CD4 + and NK T cells is abolished in STAT1 –/– and IFN-γ –/– but not in IL-6 –/– mice. Wild-type and knockout mice were injected with Con A for 3, 6, and 9 hours. Hepatic lymphocytes were isolated. The surface of CD4 + CD69 + or NK1.1 + CD3 + was analyzed by flow cytometry. The flow cytometric analysis is representative of three independent experiments. The upper-right quadrant in each panel shows CD3 + CD69 + or NK1.1 + CD3 + double-positive cells (percentage of the total hepatic lymphocytes). Values are shown in d as means ± SEM from three mice at each time point. * P
    Figure Legend Snippet: Con A injection–mediated activation of CD4 + and NK T cells is abolished in STAT1 –/– and IFN-γ –/– but not in IL-6 –/– mice. Wild-type and knockout mice were injected with Con A for 3, 6, and 9 hours. Hepatic lymphocytes were isolated. The surface of CD4 + CD69 + or NK1.1 + CD3 + was analyzed by flow cytometry. The flow cytometric analysis is representative of three independent experiments. The upper-right quadrant in each panel shows CD3 + CD69 + or NK1.1 + CD3 + double-positive cells (percentage of the total hepatic lymphocytes). Values are shown in d as means ± SEM from three mice at each time point. * P

    Techniques Used: Injection, Activation Assay, Mouse Assay, Knock-Out, Isolation, Flow Cytometry, Cytometry

    29) Product Images from "Hepatitis B Core Antigen Impairs the Polarization While Promoting the Production of Inflammatory Cytokines of M2 Macrophages via the TLR2 Pathway"

    Article Title: Hepatitis B Core Antigen Impairs the Polarization While Promoting the Production of Inflammatory Cytokines of M2 Macrophages via the TLR2 Pathway

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00535

    HBcAg retrieved the production of inflammatory cytokines by M2 macrophages and inhibited M2 phenotype correlated with STAT6 pathway. (A,B) After being stimulated with GM-CSF or M-CSF for 6 d, M0–M1 and M0–M2 unpolarized MΦ were incubated with LPS/IFN-γ or IL-4 for further differentiation. After another 24 h incubation, polarized M1 or M2 MΦ were stimulated with 10 μg/ml HBcAg or an equal volume of PBS for another 30 min. The protein levels of STAT1, p-STAT1, STAT6, and p-STAT6 were analyzed by Western blot. (A) Representative figures and (B) cumulative densitometry quantification for p-STAT1 and p-STAT6 expression normalized to STAT1 and STAT6, respectively. (C) Supernatants of polarized M1 and M2 MΦ stimulated with HBcAg or PBS were collected to measure TNF-α, IL-6, and IL-10 productions by ELISA. Experiments in (A,B) were repeated at least three times. Data were analyzed using a paired Student's t -test. Results are shown as the mean ± SEM ( A,B , n = 3; C , n = 9–11). * p
    Figure Legend Snippet: HBcAg retrieved the production of inflammatory cytokines by M2 macrophages and inhibited M2 phenotype correlated with STAT6 pathway. (A,B) After being stimulated with GM-CSF or M-CSF for 6 d, M0–M1 and M0–M2 unpolarized MΦ were incubated with LPS/IFN-γ or IL-4 for further differentiation. After another 24 h incubation, polarized M1 or M2 MΦ were stimulated with 10 μg/ml HBcAg or an equal volume of PBS for another 30 min. The protein levels of STAT1, p-STAT1, STAT6, and p-STAT6 were analyzed by Western blot. (A) Representative figures and (B) cumulative densitometry quantification for p-STAT1 and p-STAT6 expression normalized to STAT1 and STAT6, respectively. (C) Supernatants of polarized M1 and M2 MΦ stimulated with HBcAg or PBS were collected to measure TNF-α, IL-6, and IL-10 productions by ELISA. Experiments in (A,B) were repeated at least three times. Data were analyzed using a paired Student's t -test. Results are shown as the mean ± SEM ( A,B , n = 3; C , n = 9–11). * p

    Techniques Used: Incubation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

    30) Product Images from "Aneuploid cells activate NF-κB to promote their immune clearance by NK cells"

    Article Title: Aneuploid cells activate NF-κB to promote their immune clearance by NK cells

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.172239

    STAT1 inactivation does not affect NK cells-mediated killing of aneuploid cells. (A) STAT1 KO RPE1-hTERT cells were generated using CRISPR-Cas9 method and single cell clones were obtained. Aneuploid cycling (A.Cyc.) and ArCK cells lacking STAT1 were generated as described in Figure 1A and E . NK cell-mediated cell death was compared to control single cell clone which harbors an empty vector as described in Figure 1B (n=3; mean ± SD; n.s -not significant). The behavior of three different clones is shown in figure.
    Figure Legend Snippet: STAT1 inactivation does not affect NK cells-mediated killing of aneuploid cells. (A) STAT1 KO RPE1-hTERT cells were generated using CRISPR-Cas9 method and single cell clones were obtained. Aneuploid cycling (A.Cyc.) and ArCK cells lacking STAT1 were generated as described in Figure 1A and E . NK cell-mediated cell death was compared to control single cell clone which harbors an empty vector as described in Figure 1B (n=3; mean ± SD; n.s -not significant). The behavior of three different clones is shown in figure.

    Techniques Used: Generated, CRISPR, Clone Assay, Plasmid Preparation

    STAT1-responsive genes are induced in aneuploid cells. (A) Significantly differentially expressed STAT1 target genes in ArCK cells compared to euploid control cells (Log2 fold change, p value
    Figure Legend Snippet: STAT1-responsive genes are induced in aneuploid cells. (A) Significantly differentially expressed STAT1 target genes in ArCK cells compared to euploid control cells (Log2 fold change, p value

    Techniques Used:

    31) Product Images from "Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells"

    Article Title: Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA118.000735

    Validation of altered proteins by immunoblotting assay. SDS-PAGE was performed on treated and untreated lysates, followed by membrane transfer and incubation with anti-OAS3, anti-SAMHD1, anti-STAT1, and anti-GAPDH overnight. The proteins of interest were visualized using the LI-COR Odyssey system. Consistent with proteomic results, the expression of OAS3, SAMHD1 and STAT1 increased after IFN-λ3 treatment. These experiments were performed in triplicate.
    Figure Legend Snippet: Validation of altered proteins by immunoblotting assay. SDS-PAGE was performed on treated and untreated lysates, followed by membrane transfer and incubation with anti-OAS3, anti-SAMHD1, anti-STAT1, and anti-GAPDH overnight. The proteins of interest were visualized using the LI-COR Odyssey system. Consistent with proteomic results, the expression of OAS3, SAMHD1 and STAT1 increased after IFN-λ3 treatment. These experiments were performed in triplicate.

    Techniques Used: SDS Page, Incubation, Expressing

    32) Product Images from "IFN?/TNF? synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response"

    Article Title: IFN?/TNF? synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

    Journal: Cell Research

    doi: 10.1038/cr.2013.47

    DUOX2 induction is regulated in a STAT2/IRF9-dependent, STAT1-independent manner. (A – C) A549 cells were transfected with siRNA specific for STAT1, STAT2 or IRF9. Forty eight h post-transfection, cells were stimulated with SN-SeV-UV for 24 h in
    Figure Legend Snippet: DUOX2 induction is regulated in a STAT2/IRF9-dependent, STAT1-independent manner. (A – C) A549 cells were transfected with siRNA specific for STAT1, STAT2 or IRF9. Forty eight h post-transfection, cells were stimulated with SN-SeV-UV for 24 h in

    Techniques Used: Transfection

    33) Product Images from "Salmonella Protein AvrA Activates the STAT3 Signaling Pathway in Colon Cancer"

    Article Title: Salmonella Protein AvrA Activates the STAT3 Signaling Pathway in Colon Cancer

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2016.04.001

    AvrA modulates STAT3 activation in AOM/DSS colonic tumors. (A, left panel) Representative Western blots of STAT1/3 and p-STAT1/3 in control colons and tumors. (Right panel) Quantitation of nuclear pSAT3. (B and C) Immunostaining of p-STAT3 and STAT3 in control colons and tumors from indicated groups. (D) Quantitation of nuclear p-STAT3 in normal and tumor tissue in the indicated groups. Data are means ± SD. n = 5 mice/group. * P
    Figure Legend Snippet: AvrA modulates STAT3 activation in AOM/DSS colonic tumors. (A, left panel) Representative Western blots of STAT1/3 and p-STAT1/3 in control colons and tumors. (Right panel) Quantitation of nuclear pSAT3. (B and C) Immunostaining of p-STAT3 and STAT3 in control colons and tumors from indicated groups. (D) Quantitation of nuclear p-STAT3 in normal and tumor tissue in the indicated groups. Data are means ± SD. n = 5 mice/group. * P

    Techniques Used: Activation Assay, Western Blot, Quantitation Assay, Immunostaining, Mouse Assay

    34) Product Images from "Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling"

    Article Title: Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156496

    USP18 silencing increases the antiviral activity of IFN-α associated with the JAK-STAT signaling pathway. (A) Hepg2.2.15 cells were treated with shRR or shUSP18 for 48 hours and then subjected to immunofluorescence analysis using a STAT1-specific polyclonal antibody. Blue, DAPI; green, GFP; red, anti-STAT1; original magnification × 400. Hepg2.2.15 cells were treated with indicated concentration of IFN-α (0, 1, 10, 100, 1000 IU/ml) for 20 hours after either shUSP18 or shRR lentivirus transduction. (B) The protein levels of p-STAT1 and STAT1 were quantified by western blot. (C) STAT1 activation was initiated and prolonged in USP18-silenced HepG2.2.15 cells and STAT1 phosphorylation remained 24 hours after treatment compared to the control group.
    Figure Legend Snippet: USP18 silencing increases the antiviral activity of IFN-α associated with the JAK-STAT signaling pathway. (A) Hepg2.2.15 cells were treated with shRR or shUSP18 for 48 hours and then subjected to immunofluorescence analysis using a STAT1-specific polyclonal antibody. Blue, DAPI; green, GFP; red, anti-STAT1; original magnification × 400. Hepg2.2.15 cells were treated with indicated concentration of IFN-α (0, 1, 10, 100, 1000 IU/ml) for 20 hours after either shUSP18 or shRR lentivirus transduction. (B) The protein levels of p-STAT1 and STAT1 were quantified by western blot. (C) STAT1 activation was initiated and prolonged in USP18-silenced HepG2.2.15 cells and STAT1 phosphorylation remained 24 hours after treatment compared to the control group.

    Techniques Used: Activity Assay, Immunofluorescence, Concentration Assay, Transduction, Western Blot, Activation Assay

    35) Product Images from "Exogenous Gamma and Alpha/Beta Interferon Rescues Human Macrophages from Cell Death Induced by Bacillus anthracis"

    Article Title: Exogenous Gamma and Alpha/Beta Interferon Rescues Human Macrophages from Cell Death Induced by Bacillus anthracis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.3.1291-1297.2004

    B. anthracis inhibits IFN-α/β signaling. Human macrophages were incubated with the Sterne strain (34F 2 ) with either IFN-β at 1,000 U/ml, IFN-γ at 400 U/ml, or saline at an MOI of 20:1 for 4.5 h. (A) Immunoblot for p tyr701 -STAT1 and total STAT1. Infection of human AM with 34F 2 failed to induce p tyr701 -STAT1 at 4.5 h (compare lanes 1 and 4) and reduced the degree of STAT1 phosphorylation by IFN-β twofold compared to uninfected IFN-treated controls (compare lanes 3 and 6). 34F 2 had a minimal effect on IFN-γ-induced p tyr701 -STAT1 formation (compare lanes 2 and 5). (B) Immunoblot for p ser727 -STAT1 and total STAT1 from STAT1-immunoprecipitated cell lysates. Infection of human THP-1 macrophages with 34F 2 failed to induce p ser727 -STAT1 at 4.5 h (compare lanes 1 and 4) and reduced the degree of STAT1 phosphorylation by IFN-β twofold compared to uninfected IFN-treated controls (compare lanes 3 and 6). 34F 2 had a minimal effect on IFN-γ-induced p ser727 -STAT1 formation (compare lanes 2 and 5). (C) EMSA for ISGF-3. Infection of human AM with 34F 2 failed to induce ISGF-3 DNA binding at 4.5 h (compare lanes 1 and 3) and inhibited IFN-β-induced ISGF-3 production (compare lanes 2 and 4). This band was confirmed to be ISGF-3 in a separate experiment by both cold competition (lane 6) and supershift with antibodies to STAT1, STAT2, and IRF-9 (lanes 7 to 9). (D) EMSA for STAT1 homodimer (GAS binding). IFN-γ induced STAT1 homodimer formation (lane 2), which was unaffected by infection of human AM with 34F 2 (lane 5). This complex was supershifted with STAT1 antibody (lane 3). In all cases lanes were normalized for total protein.
    Figure Legend Snippet: B. anthracis inhibits IFN-α/β signaling. Human macrophages were incubated with the Sterne strain (34F 2 ) with either IFN-β at 1,000 U/ml, IFN-γ at 400 U/ml, or saline at an MOI of 20:1 for 4.5 h. (A) Immunoblot for p tyr701 -STAT1 and total STAT1. Infection of human AM with 34F 2 failed to induce p tyr701 -STAT1 at 4.5 h (compare lanes 1 and 4) and reduced the degree of STAT1 phosphorylation by IFN-β twofold compared to uninfected IFN-treated controls (compare lanes 3 and 6). 34F 2 had a minimal effect on IFN-γ-induced p tyr701 -STAT1 formation (compare lanes 2 and 5). (B) Immunoblot for p ser727 -STAT1 and total STAT1 from STAT1-immunoprecipitated cell lysates. Infection of human THP-1 macrophages with 34F 2 failed to induce p ser727 -STAT1 at 4.5 h (compare lanes 1 and 4) and reduced the degree of STAT1 phosphorylation by IFN-β twofold compared to uninfected IFN-treated controls (compare lanes 3 and 6). 34F 2 had a minimal effect on IFN-γ-induced p ser727 -STAT1 formation (compare lanes 2 and 5). (C) EMSA for ISGF-3. Infection of human AM with 34F 2 failed to induce ISGF-3 DNA binding at 4.5 h (compare lanes 1 and 3) and inhibited IFN-β-induced ISGF-3 production (compare lanes 2 and 4). This band was confirmed to be ISGF-3 in a separate experiment by both cold competition (lane 6) and supershift with antibodies to STAT1, STAT2, and IRF-9 (lanes 7 to 9). (D) EMSA for STAT1 homodimer (GAS binding). IFN-γ induced STAT1 homodimer formation (lane 2), which was unaffected by infection of human AM with 34F 2 (lane 5). This complex was supershifted with STAT1 antibody (lane 3). In all cases lanes were normalized for total protein.

    Techniques Used: Incubation, Infection, Immunoprecipitation, Binding Assay

    36) Product Images from "Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells"

    Article Title: Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00268

    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P
    Figure Legend Snippet: DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Techniques Used: Expressing, Quantitative RT-PCR, Infection

    37) Product Images from "Interferon-α Induces Neurotoxicity Through Activation of the Type I Receptor and the GluN2A Subunit of the NMDA Receptor"

    Article Title: Interferon-α Induces Neurotoxicity Through Activation of the Type I Receptor and the GluN2A Subunit of the NMDA Receptor

    Journal: Journal of Interferon & Cytokine Research

    doi: 10.1089/jir.2014.0105

    STAT1 is phosphorylated in neurons treated with IFNα. Protein extracts from neurons treated with IFNα at various time points were analyzed by immunoblotting for STAT1 and phospho-STAT1 (p-STAT1) expression. p-STAT1 peaked at 20 min
    Figure Legend Snippet: STAT1 is phosphorylated in neurons treated with IFNα. Protein extracts from neurons treated with IFNα at various time points were analyzed by immunoblotting for STAT1 and phospho-STAT1 (p-STAT1) expression. p-STAT1 peaked at 20 min

    Techniques Used: Expressing

    38) Product Images from "Hepatitis C virus core protein-induced miR-93-5p up-regulation inhibits interferon signaling pathway by targeting IFNAR1"

    Article Title: Hepatitis C virus core protein-induced miR-93-5p up-regulation inhibits interferon signaling pathway by targeting IFNAR1

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v24.i2.226

    The miR-93-5p-IFNAR1 axis regulates the interferon signaling pathway. A: Western blot shows the protein expression of IFNAR1 in Huh7 cells transfected with NC (200 ng/mL), siRNA (200 ng/mL), vector (200 ng/mL), or IFNAR1 (200 ng/mL). This experiment was repeated twice; B and C: Western blot shows the phosphorylation level of STAT1 and the protein expression of STAT1 in Huh7 cells transfected with NC (200 ng/mL), siRNA (200 ng/mL), vector (200 ng/mL), or IFNAR1 (200 ng/mL). This experiment was repeated twice; D and E: Western blot shows the phosphorylation level of STAT1 and the protein expression of STAT1 in Huh7 cells transfected with ago-NC (200 nmol/L), miR-93-5p agomir (200 nmol/L), antago-NC (200 nmol/L), or miR-93-5p antagomir (200 nmol/L). This experiment was repeated twice; F and G: Western blot shows the phosphorylation level of STAT1 and the protein expression of STAT1 and IFNAR1 in Huh7 cells transfected with ago-NC (200 nmol/L), miR-93-5p agomir (200 nmol/L), or miR-93-5p agomir (200 nmol/L) plus IFNAR1 (200 ng/mL). IFNAR1: Interferon receptor 1.
    Figure Legend Snippet: The miR-93-5p-IFNAR1 axis regulates the interferon signaling pathway. A: Western blot shows the protein expression of IFNAR1 in Huh7 cells transfected with NC (200 ng/mL), siRNA (200 ng/mL), vector (200 ng/mL), or IFNAR1 (200 ng/mL). This experiment was repeated twice; B and C: Western blot shows the phosphorylation level of STAT1 and the protein expression of STAT1 in Huh7 cells transfected with NC (200 ng/mL), siRNA (200 ng/mL), vector (200 ng/mL), or IFNAR1 (200 ng/mL). This experiment was repeated twice; D and E: Western blot shows the phosphorylation level of STAT1 and the protein expression of STAT1 in Huh7 cells transfected with ago-NC (200 nmol/L), miR-93-5p agomir (200 nmol/L), antago-NC (200 nmol/L), or miR-93-5p antagomir (200 nmol/L). This experiment was repeated twice; F and G: Western blot shows the phosphorylation level of STAT1 and the protein expression of STAT1 and IFNAR1 in Huh7 cells transfected with ago-NC (200 nmol/L), miR-93-5p agomir (200 nmol/L), or miR-93-5p agomir (200 nmol/L) plus IFNAR1 (200 ng/mL). IFNAR1: Interferon receptor 1.

    Techniques Used: Western Blot, Expressing, Transfection, Plasmid Preparation

    Hepatitis C virus-1b core protein increases miR-93-5p expression and inactivates the interferon signaling pathway. A: HCV core protein is enforcedly expressed in Huh7 cells using pcDNA3.1 (+) vector. Western blot shows HCV core protein expression. This experiment was repeated twice; B: Bar shows miR-122 expression in Huh7 cells transfected with pcDNA 3.1 (+) empty vector or HCV core-pcDNA3.1 (+) vector. U6 was used as an internal reference. This experiment was repeated three times. C: Bar shows miR-93-5p expression in Huh7 cells transfected with pcDNA 3.1 (+) empty vector or HCV core-pcDNA3.1 (+) vector. U6 was used as an internal reference. This experiment was repeated three times. D and E: Western blot shows the protein expression of IFNAR1 and STAT1, as well as the phosphorylation level of STAT1 in Huh7 cells which were transfected with pcDNA 3.1 (+) empty vector or HCV core-pcDNA3.1 (+) vector. This experiment was repeated twice. HCV: Hepatitis C virus; IFNAR1: Interferon receptor 1.
    Figure Legend Snippet: Hepatitis C virus-1b core protein increases miR-93-5p expression and inactivates the interferon signaling pathway. A: HCV core protein is enforcedly expressed in Huh7 cells using pcDNA3.1 (+) vector. Western blot shows HCV core protein expression. This experiment was repeated twice; B: Bar shows miR-122 expression in Huh7 cells transfected with pcDNA 3.1 (+) empty vector or HCV core-pcDNA3.1 (+) vector. U6 was used as an internal reference. This experiment was repeated three times. C: Bar shows miR-93-5p expression in Huh7 cells transfected with pcDNA 3.1 (+) empty vector or HCV core-pcDNA3.1 (+) vector. U6 was used as an internal reference. This experiment was repeated three times. D and E: Western blot shows the protein expression of IFNAR1 and STAT1, as well as the phosphorylation level of STAT1 in Huh7 cells which were transfected with pcDNA 3.1 (+) empty vector or HCV core-pcDNA3.1 (+) vector. This experiment was repeated twice. HCV: Hepatitis C virus; IFNAR1: Interferon receptor 1.

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Transfection

    39) Product Images from "Severe traumatic hemorrhagic shock induces compromised immune barrier function of the mesenteric lymph node leading to an increase in intestinal bacterial translocation"

    Article Title: Severe traumatic hemorrhagic shock induces compromised immune barrier function of the mesenteric lymph node leading to an increase in intestinal bacterial translocation

    Journal: American Journal of Translational Research

    doi:

    Polarization of naïve T cells induced by mesenteric lymph node dendritic cells. Naïve CD4 + T cells (1×10 6 cells/well) were co-cultured with OX62 + MLN-DCs (1×10 5 cells/well) for 3 days at 37°C with 5% CO 2 in a humidified incubator. Cells were then analyzed by flow cytometry for Th1(CD4 + IFNγ + ) (A, upper), Th2(CD4 + IL-4 + ) (A, lower), and Treg (gated on CD4 + , CD25 + FOXP3 + ) (E). Summary statistics are shown for Th1 (B), Th2 (C), Th1/Th2 (D), and Treg (F). A Western blot shows the expression of STAT1, STAT6, and their phosphorylated forms in polarized CD4 + T cells from different groups (G). Data are expressed as mean ± SD, and there were n = 10 animals per group. In (B, C and F) ***P
    Figure Legend Snippet: Polarization of naïve T cells induced by mesenteric lymph node dendritic cells. Naïve CD4 + T cells (1×10 6 cells/well) were co-cultured with OX62 + MLN-DCs (1×10 5 cells/well) for 3 days at 37°C with 5% CO 2 in a humidified incubator. Cells were then analyzed by flow cytometry for Th1(CD4 + IFNγ + ) (A, upper), Th2(CD4 + IL-4 + ) (A, lower), and Treg (gated on CD4 + , CD25 + FOXP3 + ) (E). Summary statistics are shown for Th1 (B), Th2 (C), Th1/Th2 (D), and Treg (F). A Western blot shows the expression of STAT1, STAT6, and their phosphorylated forms in polarized CD4 + T cells from different groups (G). Data are expressed as mean ± SD, and there were n = 10 animals per group. In (B, C and F) ***P

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Western Blot, Expressing

    40) Product Images from "Rubicon Modulates Antiviral Type I Interferon (IFN) Signaling by Targeting IFN Regulatory Factor 3 Dimerization"

    Article Title: Rubicon Modulates Antiviral Type I Interferon (IFN) Signaling by Targeting IFN Regulatory Factor 3 Dimerization

    Journal: Journal of Virology

    doi: 10.1128/JVI.00248-17

    Rubicon inhibits the type I IFN signaling pathway and suppresses expression of IFN-related genes. (A and B) Control RAW264.7 (RAW-Scramble) and Rubicon knockdown RAW264.7 (RAW-sh-Rubicon) cells (A) or control RAW264.7 (RAW-Control) and Rubicon-overexpressing RAW264.7 (RAW-Rubicon) cells (B) were infected with PR8-GFP (MOI = 2). At the indicated times after infection, IRF3, p65, and STAT1 and phosphorylated IRF3, p65, STAT1, p38, and TBK1 in cell extracts were measured by immunoblotting. β-Actin was used to confirm equal loading of proteins. (C) Total RNAs were extracted from RAW-Scramble and RAW-sh-Rubicon cells after PR8-GFP (MOI = 3) infection. Expression of the indicated mRNA-encoding genes was analyzed by real time quantitative reverse transcription-PCR (qRT-PCR) at 0, 4, 8, 12, 16, and 24 hpi (IFN) or at 0 and 16 hpi (IFN-α, PKR, OAS, GBP1, MX1, PML, ISG-15, ISG-20, p56, and IL-6). The data are expressed as means and SEM and are representative of the results of at least two independent experiments.
    Figure Legend Snippet: Rubicon inhibits the type I IFN signaling pathway and suppresses expression of IFN-related genes. (A and B) Control RAW264.7 (RAW-Scramble) and Rubicon knockdown RAW264.7 (RAW-sh-Rubicon) cells (A) or control RAW264.7 (RAW-Control) and Rubicon-overexpressing RAW264.7 (RAW-Rubicon) cells (B) were infected with PR8-GFP (MOI = 2). At the indicated times after infection, IRF3, p65, and STAT1 and phosphorylated IRF3, p65, STAT1, p38, and TBK1 in cell extracts were measured by immunoblotting. β-Actin was used to confirm equal loading of proteins. (C) Total RNAs were extracted from RAW-Scramble and RAW-sh-Rubicon cells after PR8-GFP (MOI = 3) infection. Expression of the indicated mRNA-encoding genes was analyzed by real time quantitative reverse transcription-PCR (qRT-PCR) at 0, 4, 8, 12, 16, and 24 hpi (IFN) or at 0 and 16 hpi (IFN-α, PKR, OAS, GBP1, MX1, PML, ISG-15, ISG-20, p56, and IL-6). The data are expressed as means and SEM and are representative of the results of at least two independent experiments.

    Techniques Used: Expressing, Infection, Polymerase Chain Reaction, Quantitative RT-PCR

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    Article Snippet: .. The cell lysates were subjected to immunoprecipitation and immunoblot as previously described ( ) using antibodies of phospho-STAT1 (pSTAT1) (Tyr-701), pSTAT3 (Tyr-705), STAT1, and STAT3 (Cell Signaling Technology, Inc., Danvers, MA), MKP-1 and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and iNOS, (BD Biosciences, San Jose, CA). .. For quantitation of the changes in STAT1 phosphorylation, the intensities of the bands representing STAT1 and pSTAT1 (Tyr-701) were measured by densitometry using an image scanner (EPSON GT-8000) and National Institutes of Health Image software.

    other:

    Article Title: MiR-155 Induction by Microbes/Microbial Ligands Requires NF-?B-Dependent de novo Protein Synthesis
    Article Snippet: Antibodies Antibodies against phospho-Stat1, c-Fos, and c-Jun were purchased from Cell Signaling (Beverly, Massachusetts).

    Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization
    Article Snippet: The primary antibodies against p-JNK (Thr183/Tyr185), JNK, p-p38 (Thr180/Tyr182), p38, p-STAT1 (Ser727), STAT1, p-ERK (Thr202/Tyr204), ERK, p-p65 (Ser536), p65 and H3 were purchased from Cell Signaling Technology.

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    Effect of cPTIO and GSH on LNO 2 - and OA-NO 2 -mediated inhibition of STAT phosphorylation. A, Role of exogenous NO release on LPS-induced <t>STAT1</t> phosphorylation. Quiescent RAW264.7 cells were simultaneously treated with various stimuli for 2 h as indicated. The cell lysates were subjected to immunoblot analysis using antibodies to <t>pSTAT1</t> (Tyr-701), STAT1, and GAPDH. Results are representative of at least three separate experiments. B, Effect of GSH quench on the inhibitory effect of LNO 2 and OA-NO 2 on LPS-induced STAT1 phosphorylation. Quiescent RAW264.7 cells were simultaneously treated with various stimuli for 2 h as indicated. Western blot was performed as described above. Results are representative of at least three separate experiments.
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    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 <t>(STAT1),</t> and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P
    Anti Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti stat1 antibody
    Activation <t>STAT1</t> and STAT3 by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 8 hours. After treatment, STAT1 ((a) N =3 replicates per condition from three independent experiments) and STAT3 phosphorylation ((b) N =3 replicates per condition from three independent experiments) were evaluated by western blot analysis. These phosphorylation levels were normalised based on β-actin levels. Whether IFN-γ induces translocalisation of STAT1 and STAT3 into nucleus was evaluated. MCs were treated with 5 ng/mL IFN-γ for 8 hours. Thereafter, localisations of STAT1 and STAT3 were determined by immunofluorescence staining ((c) and (d)). Data are expressed as mean ± SE. Asterisk indicates a significant difference compared with the control group at each time point ( P
    Rabbit Anti Stat1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti stat1 antibody/product/Cell Signaling Technology Inc
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    99
    Cell Signaling Technology Inc rabbit anti phospho stat1 tyr701 monoclonal antibody
    Species-specific activity of rec Mg IFN-γ and mouse IFN-γ on different rodent cell lines. NIH/3T3, BVK168, FMN-R and AAL-R cells were tested and either left untreated as control (lower panel), stimulated for 1 h with rec Mg IFN-γ (200 ng/ml, upper panel) or mouse IFN-γ (200 U/ml, central panel). In each panel the following stainings are shown from left to right: <t>phospho-Tyr701-STAT1</t> staining (green), DAPI staining (white) and overlay of the two signals. Activated phospho-STAT1 is indicated by nuclear translocation. Of note, minor background signal in the cytoplasma was observed in some control treated cells. A representative experiment of three replicates is shown. The scale bar represents 50 μm.
    Rabbit Anti Phospho Stat1 Tyr701 Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho stat1 tyr701 monoclonal antibody/product/Cell Signaling Technology Inc
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    Price from $9.99 to $1999.99
    rabbit anti phospho stat1 tyr701 monoclonal antibody - by Bioz Stars, 2020-11
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    Image Search Results


    Effect of cPTIO and GSH on LNO 2 - and OA-NO 2 -mediated inhibition of STAT phosphorylation. A, Role of exogenous NO release on LPS-induced STAT1 phosphorylation. Quiescent RAW264.7 cells were simultaneously treated with various stimuli for 2 h as indicated. The cell lysates were subjected to immunoblot analysis using antibodies to pSTAT1 (Tyr-701), STAT1, and GAPDH. Results are representative of at least three separate experiments. B, Effect of GSH quench on the inhibitory effect of LNO 2 and OA-NO 2 on LPS-induced STAT1 phosphorylation. Quiescent RAW264.7 cells were simultaneously treated with various stimuli for 2 h as indicated. Western blot was performed as described above. Results are representative of at least three separate experiments.

    Journal: Endocrinology

    Article Title: Nitroalkenes Suppress Lipopolysaccharide-Induced Signal Transducer and Activator of Transcription Signaling in Macrophages: A Critical Role of Mitogen-Activated Protein Kinase Phosphatase 1

    doi: 10.1210/en.2007-1639

    Figure Lengend Snippet: Effect of cPTIO and GSH on LNO 2 - and OA-NO 2 -mediated inhibition of STAT phosphorylation. A, Role of exogenous NO release on LPS-induced STAT1 phosphorylation. Quiescent RAW264.7 cells were simultaneously treated with various stimuli for 2 h as indicated. The cell lysates were subjected to immunoblot analysis using antibodies to pSTAT1 (Tyr-701), STAT1, and GAPDH. Results are representative of at least three separate experiments. B, Effect of GSH quench on the inhibitory effect of LNO 2 and OA-NO 2 on LPS-induced STAT1 phosphorylation. Quiescent RAW264.7 cells were simultaneously treated with various stimuli for 2 h as indicated. Western blot was performed as described above. Results are representative of at least three separate experiments.

    Article Snippet: The cell lysates were subjected to immunoprecipitation and immunoblot as previously described ( ) using antibodies of phospho-STAT1 (pSTAT1) (Tyr-701), pSTAT3 (Tyr-705), STAT1, and STAT3 (Cell Signaling Technology, Inc., Danvers, MA), MKP-1 and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and iNOS, (BD Biosciences, San Jose, CA).

    Techniques: Inhibition, Western Blot

    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells

    doi: 10.3389/fcimb.2018.00268

    Figure Lengend Snippet: DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Article Snippet: The following primary antibodies were used: anti-caspase 1 (GTX111630, GeneTex, Irvine, CA), anti-caspase 3 (#9665, Cell Signaling Technology, Danvers, MA), anti-caspase 7 (GTX1002337, GeneTex), anti-caspase 8 (#4790, Cell Signaling Technology), anti-bone morphogenetic protein 4 (BMP-4; GTX100875, GeneTex), anti-phospho-STAT1 (phospho-Tyr701, #9167 Cell signaling), anti-STAT1 (#14994, Cell Signaling), anti-phospho-STAT2 (phospho-Tyr690, GTX50721, GeneTex), anti-STAT2 (#14994, Cell Signaling), anti-DENV NS3 (GTX124252, GeneTex), and anti-GAPDH (#60004-1-Ig, Proteintech Group, Rosemont, IL).

    Techniques: Expressing, Quantitative RT-PCR, Infection

    Activation STAT1 and STAT3 by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 8 hours. After treatment, STAT1 ((a) N =3 replicates per condition from three independent experiments) and STAT3 phosphorylation ((b) N =3 replicates per condition from three independent experiments) were evaluated by western blot analysis. These phosphorylation levels were normalised based on β-actin levels. Whether IFN-γ induces translocalisation of STAT1 and STAT3 into nucleus was evaluated. MCs were treated with 5 ng/mL IFN-γ for 8 hours. Thereafter, localisations of STAT1 and STAT3 were determined by immunofluorescence staining ((c) and (d)). Data are expressed as mean ± SE. Asterisk indicates a significant difference compared with the control group at each time point ( P

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Activation STAT1 and STAT3 by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 8 hours. After treatment, STAT1 ((a) N =3 replicates per condition from three independent experiments) and STAT3 phosphorylation ((b) N =3 replicates per condition from three independent experiments) were evaluated by western blot analysis. These phosphorylation levels were normalised based on β-actin levels. Whether IFN-γ induces translocalisation of STAT1 and STAT3 into nucleus was evaluated. MCs were treated with 5 ng/mL IFN-γ for 8 hours. Thereafter, localisations of STAT1 and STAT3 were determined by immunofluorescence staining ((c) and (d)). Data are expressed as mean ± SE. Asterisk indicates a significant difference compared with the control group at each time point ( P

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining

    Contribution of STAT1 to the IFN-γ-induced AGT and MCP-1 augmentations. To elucidate further the mechanisms underlying the regulation of AGT and MCP-1 by IFN-γ, basal STAT1 expression was suppressed using STAT1-siRNA ((a) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 8 hours. AGT mRNA ((b) N =3 replicates per condition from three independent experiments) and MCP-1 mRNA ((c) N =3 replicates per condition from three independent experiments) levels were measured by qRT–PCR. AGT and MCP-1 mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Contribution of STAT1 to the IFN-γ-induced AGT and MCP-1 augmentations. To elucidate further the mechanisms underlying the regulation of AGT and MCP-1 by IFN-γ, basal STAT1 expression was suppressed using STAT1-siRNA ((a) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 8 hours. AGT mRNA ((b) N =3 replicates per condition from three independent experiments) and MCP-1 mRNA ((c) N =3 replicates per condition from three independent experiments) levels were measured by qRT–PCR. AGT and MCP-1 mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Expressing, Quantitative RT-PCR

    Schematic summary of proposed MCP-1, AGT regulation by IFN-γ-STAT1 in rat MCs. IFN-γ induces MCP-1 and SOCS1 expression directly via STAT1 in rat MCs. Conversely, STAT1-induced expression of SOCS1 attenuates IFN-γ-STAT3-mediated expression of AGT, providing feedback regulation of IFN-γ-AGT signaling in rat MCs. These results demonstrate that while IFN-γ increases both AGT and MCP-1 expression, STAT1 plays an opposing role in the regulation of each factor in MCs. MCP-1: monocyte chemoattractant protein 1; AGT: angiotensinogen; IFN-γ: interferon-γ; SOCS1: suppressor of cytokine signaling 1; MCs: mesangial cells.

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Schematic summary of proposed MCP-1, AGT regulation by IFN-γ-STAT1 in rat MCs. IFN-γ induces MCP-1 and SOCS1 expression directly via STAT1 in rat MCs. Conversely, STAT1-induced expression of SOCS1 attenuates IFN-γ-STAT3-mediated expression of AGT, providing feedback regulation of IFN-γ-AGT signaling in rat MCs. These results demonstrate that while IFN-γ increases both AGT and MCP-1 expression, STAT1 plays an opposing role in the regulation of each factor in MCs. MCP-1: monocyte chemoattractant protein 1; AGT: angiotensinogen; IFN-γ: interferon-γ; SOCS1: suppressor of cytokine signaling 1; MCs: mesangial cells.

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Expressing

    Participation of SOCS1 in IFN-γ-induced AGT augmentations. Involvement of STAT1 in SOCS1 regulation was tested using STAT1-siRNA as shown in Figure 4 (a) . Furthermore, MCs were treated with SOCS1-siRNA ((b) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 24 hours. Then, AGT mRNA levels were measured by qRT–PCR (c). AGT mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

    Article Title: STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

    doi: 10.1177/1470320320946527

    Figure Lengend Snippet: Participation of SOCS1 in IFN-γ-induced AGT augmentations. Involvement of STAT1 in SOCS1 regulation was tested using STAT1-siRNA as shown in Figure 4 (a) . Furthermore, MCs were treated with SOCS1-siRNA ((b) N =3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 24 hours. Then, AGT mRNA levels were measured by qRT–PCR (c). AGT mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk ( P

    Article Snippet: A mouse anti-phospho-STAT1 (Tyr 701) antibody, a rabbit anti-phospho-STAT3 (Tyr 705) antibody, a rabbit anti-STAT1 antibody and a rabbit anti-STAT3 antibody were obtained from Cell Signaling Technology.

    Techniques: Quantitative RT-PCR

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-21143-0

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

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

    Techniques: Activity Assay, Staining, Translocation Assay