irf1 d5e4 xp rabbit mab  (Cell Signaling Technology Inc)

 
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    Name:
    IRF 1 D5E4 XP Rabbit mAb
    Description:
    Interferon regulatory factors IRFs comprise a family of transcription factors that function within the Jak Stat pathway to regulate interferon IFN and IFN inducible gene expression in response to viral infection 1 IRFs play an important role in pathogen defense autoimmunity lymphocyte development cell growth and susceptibility to transformation The IRF family includes nine members IRF 1 IRF 2 IRF 9 ISGF3γ IRF 3 IRF 4 Pip LSIRF ICSAT IRF 5 IRF 6 IRF 7 and IRF 8 ICSBP All IRF proteins share homology in their amino terminal DNA binding domains IRF family members regulate transcription through interactions with proteins that share similar DNA binding motifs such as IFN stimulated response elements ISRE IFN consensus sequences ICS and IFN regulatory elements IRF E 2 The IRF 1 transcription factor was originally identified as a regulator of virus inducible enhancer like elements of the IFN β gene 3 IRF 1 is widely expressed and upregulated by viral infection or stimulation with IFN or other cytokines IRF 1 is serine phosphorylated by casein kinase II CKII at two clustered sites one in the DNA binding domain amino acids 138 150 and another in the transactivation domain amino acids 219 231 4 Mutation analysis of the latter site suggests that these phosphorylation sites help regulate IRF 1 activity Tyrosine phosphorylation has also been shown to be important in IFN γ mediated differentiation of myeloid cell lines 5 C terminal SUMOylated IRF 1 inhibits apoptosis in tumor cells by repression of its transcriptional activity 6
    Catalog Number:
    8478
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunohistochemistry, Immunofluorescence, Flow Cytometry
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro261 of human IRF-1 protein.
    Reactivity:
    Human Mouse Rat
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    Structured Review

    Cell Signaling Technology Inc irf1 d5e4 xp rabbit mab
    Interferon regulatory factors IRFs comprise a family of transcription factors that function within the Jak Stat pathway to regulate interferon IFN and IFN inducible gene expression in response to viral infection 1 IRFs play an important role in pathogen defense autoimmunity lymphocyte development cell growth and susceptibility to transformation The IRF family includes nine members IRF 1 IRF 2 IRF 9 ISGF3γ IRF 3 IRF 4 Pip LSIRF ICSAT IRF 5 IRF 6 IRF 7 and IRF 8 ICSBP All IRF proteins share homology in their amino terminal DNA binding domains IRF family members regulate transcription through interactions with proteins that share similar DNA binding motifs such as IFN stimulated response elements ISRE IFN consensus sequences ICS and IFN regulatory elements IRF E 2 The IRF 1 transcription factor was originally identified as a regulator of virus inducible enhancer like elements of the IFN β gene 3 IRF 1 is widely expressed and upregulated by viral infection or stimulation with IFN or other cytokines IRF 1 is serine phosphorylated by casein kinase II CKII at two clustered sites one in the DNA binding domain amino acids 138 150 and another in the transactivation domain amino acids 219 231 4 Mutation analysis of the latter site suggests that these phosphorylation sites help regulate IRF 1 activity Tyrosine phosphorylation has also been shown to be important in IFN γ mediated differentiation of myeloid cell lines 5 C terminal SUMOylated IRF 1 inhibits apoptosis in tumor cells by repression of its transcriptional activity 6
    https://www.bioz.com/result/irf1 d5e4 xp rabbit mab/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irf1 d5e4 xp rabbit mab - by Bioz Stars, 2020-09
    93/100 stars

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    Related Articles

    Immunostaining:

    Article Title: Interferon regulatory factor-1 activates autophagy to aggravate hepatic ischemia-reperfusion injury via the P38/P62 pathway in mice
    Article Snippet: .. Rabbit Abs specific for IRF-1 (8478), P38 (8690), p-P38 (4511), LC3 (12741), caspase-3 (9663), cleaved caspase-3 (9664) and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (7074) were purchased from Cell Signaling Technology Inc. Anti-P62 Abs (5114) for immunoblotting were from Cell Signaling Technology Inc and anti-P62 Abs (BA2849) for immunostaining were from Boster. .. Anti-PCNA Ab (BS5842) was from Bioworld Technology Inc. Anti-GAPDH Ab (TA-08) was from ZSGB-BIO.

    Incubation:

    Article Title: Role of interferon regulatory factor-1 in lipopolysaccharide-induced mitochondrial damage and oxidative stress responses in macrophages
    Article Snippet: .. After blocking with 5% non-fat milk in 10 mM Tris-buffered saline (pH 7.5) with 0.1% Tween-20 (TBS-T) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against rabbit monoclonal antibody of IRF-1 (1:1,000; Cat. no. 8478) and Histone H3 (1:2,000; Cat. no. 4499) (Cell Signaling Technology, Beverly, MA, USA). .. After 3 washes in TBS-T, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 1 h. After 3 washes, the blots were developed with electrochemiluminescence (ECL) (Beyotime) and visualized with a ChemiDoc MP imaging system (Bio-Rad Laboratories, Berkeley, CA, USA).

    Article Title: Brucella abortus triggers a cGAS-independent STING pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation
    Article Snippet: .. The following primary antibodies were incubated overnight at 4°C: rabbit mAb IRF-1 #8478 (Cell Signaling); rabbit mAb β-Actin #4970 (Cell Signaling) anti-Caspase-1 (p20, mouse mAb #AG-20B-0042, Adipogen) and IL-1β (mouse mAb #3A6, Cell Signaling). .. For testing siRNA knock down efficiency on hTERT cells, similar procedure was performed using rabbit anti-STING polyclonal antibody 1:5000 and rabbit anti-cGAS (#D1D3G, Cell Signaling) 1:1000.

    other:

    Article Title: Upregulation of PD-L1 expression by resveratrol and piceatannol in breast and colorectal cancer cells occurs via HDAC3/p300-mediated NF-κB signaling
    Article Snippet: The antibodies for human PD-L1 (E1L3N, 13684), p38 MAPK (D13E1, 8690), NF-κB p65 (D14E12, 8242), γH2AX (20E3, 9718), cleaved caspase 3 (D3E9, 9579), IRF-1 (D5E4, 8478) and rabbit IgG isotype monoclonal antibody (DA1E, 5742) conjugated to PE were obtained from Cell Signaling Technology (Beverly, MA, USA). c-Myc antibody (9E10, sc-40) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Blocking Assay:

    Article Title: Role of interferon regulatory factor-1 in lipopolysaccharide-induced mitochondrial damage and oxidative stress responses in macrophages
    Article Snippet: .. After blocking with 5% non-fat milk in 10 mM Tris-buffered saline (pH 7.5) with 0.1% Tween-20 (TBS-T) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against rabbit monoclonal antibody of IRF-1 (1:1,000; Cat. no. 8478) and Histone H3 (1:2,000; Cat. no. 4499) (Cell Signaling Technology, Beverly, MA, USA). .. After 3 washes in TBS-T, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 1 h. After 3 washes, the blots were developed with electrochemiluminescence (ECL) (Beyotime) and visualized with a ChemiDoc MP imaging system (Bio-Rad Laboratories, Berkeley, CA, USA).

    Expressing:

    Article Title: Nuclear IRF-1 expression as a mechanism to assess “Capability” to express PD-L1 and response to PD-1 therapy in metastatic melanoma
    Article Snippet: .. Antibody validation Antibodies for IRF-1 (CST D5E4; #8478) and PD-L1 (Spring Bioscience SP142; #M4420) were validated [ ] by migration on Western blot and subcellular localization with progressive expression. .. Upon treatment with IFNγ, melanoma cell lines upregulated IRF-1 and PD-L1 as detected by Western Blot (Fig. ) and immunofluorescence (Fig. ).

    Western Blot:

    Article Title: Nuclear IRF-1 expression as a mechanism to assess “Capability” to express PD-L1 and response to PD-1 therapy in metastatic melanoma
    Article Snippet: .. Antibody validation Antibodies for IRF-1 (CST D5E4; #8478) and PD-L1 (Spring Bioscience SP142; #M4420) were validated [ ] by migration on Western blot and subcellular localization with progressive expression. .. Upon treatment with IFNγ, melanoma cell lines upregulated IRF-1 and PD-L1 as detected by Western Blot (Fig. ) and immunofluorescence (Fig. ).

    Migration:

    Article Title: Nuclear IRF-1 expression as a mechanism to assess “Capability” to express PD-L1 and response to PD-1 therapy in metastatic melanoma
    Article Snippet: .. Antibody validation Antibodies for IRF-1 (CST D5E4; #8478) and PD-L1 (Spring Bioscience SP142; #M4420) were validated [ ] by migration on Western blot and subcellular localization with progressive expression. .. Upon treatment with IFNγ, melanoma cell lines upregulated IRF-1 and PD-L1 as detected by Western Blot (Fig. ) and immunofluorescence (Fig. ).

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  • 92
    Cell Signaling Technology Inc anti irf1 pe
    Opposite effects of <t>IRF1</t> and IRF4 on histone acetylation at Il9 locus. ( a – d ) Purified WT and ( a , b ) Irf4 −/− or ( c , d ) Irf1 −/− CD4 + T cells were cultured under Th9 conditions with or without IFN-γ. ( a , c ) ChIP analysis of histone H4 acetylation (Ac) at CNS of the Il9 gene. ( c ) Bars show fold induction of H4-Ac relative to WT Th9 cells treated with IFN-γ. ( b , d ) ChIP analysis for RNA polymerase II (pol II) occupancy at Il9 and Rpl32 genes. ( a – d ) The same chromatin was used for control ChIP experiments with control IgG. Precipitated DNA is presented relative to input (% input). Values for nonspecific binding (as determined by using control IgG) were subtracted. ( a – d ) Data are shown as mean±s.d. of combined results from two independent experiments (triplicates to quintuplets in qRT-PCR). The experiments were repeated ( b ) three or ( d ) five times with consistent results. * P
    Anti Irf1 Pe, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti irf1 pe/product/Cell Signaling Technology Inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti irf1 pe - by Bioz Stars, 2020-09
    92/100 stars
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    93
    Cell Signaling Technology Inc rabbit anti irf1
    Complement opsonization of HIV-1 suppressed <t>IRF1</t> and IRF7 but activated IRF3 signaling. DCs (1 × 10 6 /ml) were exposed to F-HIV, C-HIV, and CI-HIV at an MOI of 8 or were mock treated. mRNA and protein expression of IRF1 ( A and B ), IRF3 ( C and
    Rabbit Anti Irf1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti irf1/product/Cell Signaling Technology Inc
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rabbit anti irf1 - by Bioz Stars, 2020-09
    93/100 stars
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    93
    Cell Signaling Technology Inc anti irf1 rabbit monoclonal antibody
    ISRE induction by ChX710 is dependent on MAVS and <t>IRF1.</t> ( a ) MAVS was silenced by siRNA transfection in ISRE-luciferase reporter cells. After 48 hours of cultures, cells were stimulated with ChX710. After 24 hours of incubation, luciferase induction was determined. ( b ) Same experiment as ( a ) using STING-specific siRNA. ( c ) Same experiment as ( a ) using IRF1-specific siRNA. ( d ) Same experiment as ( a ) using IRF3-specific siRNA. ( e,f ) IRF3 phosphorylation was determined by western-blot in HEK-293T ( e ) or A549 cells ( f ) treated for 24 hours with DMSO alone or ChX710 at 25 μM or 50 μM. ( g ) Schematic model of the signaling pathways induced by ChX710. IRF3 phosphorylation is induced, but this is insufficient to activate IFN-β expression. Data represent means ± SD of three independent experiments, except for ( e ) and ( f ) that corresponds to representative results. ** P
    Anti Irf1 Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti irf1 rabbit monoclonal antibody/product/Cell Signaling Technology Inc
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    anti irf1 rabbit monoclonal antibody - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    Opposite effects of IRF1 and IRF4 on histone acetylation at Il9 locus. ( a – d ) Purified WT and ( a , b ) Irf4 −/− or ( c , d ) Irf1 −/− CD4 + T cells were cultured under Th9 conditions with or without IFN-γ. ( a , c ) ChIP analysis of histone H4 acetylation (Ac) at CNS of the Il9 gene. ( c ) Bars show fold induction of H4-Ac relative to WT Th9 cells treated with IFN-γ. ( b , d ) ChIP analysis for RNA polymerase II (pol II) occupancy at Il9 and Rpl32 genes. ( a – d ) The same chromatin was used for control ChIP experiments with control IgG. Precipitated DNA is presented relative to input (% input). Values for nonspecific binding (as determined by using control IgG) were subtracted. ( a – d ) Data are shown as mean±s.d. of combined results from two independent experiments (triplicates to quintuplets in qRT-PCR). The experiments were repeated ( b ) three or ( d ) five times with consistent results. * P

    Journal: Nature Communications

    Article Title: Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

    doi: 10.1038/ncomms15366

    Figure Lengend Snippet: Opposite effects of IRF1 and IRF4 on histone acetylation at Il9 locus. ( a – d ) Purified WT and ( a , b ) Irf4 −/− or ( c , d ) Irf1 −/− CD4 + T cells were cultured under Th9 conditions with or without IFN-γ. ( a , c ) ChIP analysis of histone H4 acetylation (Ac) at CNS of the Il9 gene. ( c ) Bars show fold induction of H4-Ac relative to WT Th9 cells treated with IFN-γ. ( b , d ) ChIP analysis for RNA polymerase II (pol II) occupancy at Il9 and Rpl32 genes. ( a – d ) The same chromatin was used for control ChIP experiments with control IgG. Precipitated DNA is presented relative to input (% input). Values for nonspecific binding (as determined by using control IgG) were subtracted. ( a – d ) Data are shown as mean±s.d. of combined results from two independent experiments (triplicates to quintuplets in qRT-PCR). The experiments were repeated ( b ) three or ( d ) five times with consistent results. * P

    Article Snippet: For intracellular staining of transcription factors after differentiation, cells were fixed with a Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Dilutent (eBioscience) and then stained with: anti-Foxp3-PE (FJK-16, eBioscience), anti-T-bet-PE (eBio4B10, eBioscience), anti-GATA-3-eF660 (TWAJ, eBioscience), anti-IRF4-AIFI647, (3E4, eBioscience) or anti-IRF1-PE (D5E4, Cell Signaling) mAbs.

    Techniques: Purification, Cell Culture, Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR

    Increased IRF1 binding correlates with decreased IRF4 binding at the Il9 locus. ( a – c ) Luciferase reporter assay of Th0-polarized cells transiently transfected with constant amounts of Il9 promoter-luciferase vector and with either empty vector (−), IRF4-expressing vector (IRF4) and/or: ( a ) constant amounts of IRF1-expressing vector (IRF1), ( b ) increasing amounts of IRF1 (0, 0.5, 1, 2 μg) or ( c ) IRF1 or an IRF1 mutant lacking the DNA-binding domain (ΔDBD). ( d , e , g , h ) ChIP-Seq analyses of Th9 cells with or without IFN-γ treatment. Cells were crosslinked with formaldehyde and immunoprecipitated with the indicated antibodies. Massive parallel sequencing of immunoprecipitated DNA was performed. ( d ) Numbers in Venn diagram indicate the IRF4-binding peaks. ( e ) IRF4 binding in Th9 cells and to the right IRF1 binding in Th9 cell treated with IFN-γ were analysed. Over-represented motifs shown as PWM (positional weight matrix) revealed by MEME/DREME analysis are shown. Associated piecharts indicate the frequency of designated motifs. ( f ) Non-polarized Th0 cells were transfected with either WT or the mutated M1 or M3 Il9 promoter-luciferase vectors (substitution mutation within the Il9 promoter IRF site is depicted above) together with empty vectors and/or IRF4. ( g ) ChIP-Seq analyses show IRF4 (upper lanes), IRF1 (middle lane) or H3K27-Ac (bottom two lanes) abundance. Below in grey boxes, potential IRF-binding sites are depicted including their distance from the Il9 transcription start site. ( h ) Normalized read counts from IRF4-ChIP-Seq in Th9 cells in the absence or presence of IFN-γ for CNS0, 1 and 2 are shown. ( a – c , f ) Data are combined from three independent experiments and show mean±s.d. * P

    Journal: Nature Communications

    Article Title: Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

    doi: 10.1038/ncomms15366

    Figure Lengend Snippet: Increased IRF1 binding correlates with decreased IRF4 binding at the Il9 locus. ( a – c ) Luciferase reporter assay of Th0-polarized cells transiently transfected with constant amounts of Il9 promoter-luciferase vector and with either empty vector (−), IRF4-expressing vector (IRF4) and/or: ( a ) constant amounts of IRF1-expressing vector (IRF1), ( b ) increasing amounts of IRF1 (0, 0.5, 1, 2 μg) or ( c ) IRF1 or an IRF1 mutant lacking the DNA-binding domain (ΔDBD). ( d , e , g , h ) ChIP-Seq analyses of Th9 cells with or without IFN-γ treatment. Cells were crosslinked with formaldehyde and immunoprecipitated with the indicated antibodies. Massive parallel sequencing of immunoprecipitated DNA was performed. ( d ) Numbers in Venn diagram indicate the IRF4-binding peaks. ( e ) IRF4 binding in Th9 cells and to the right IRF1 binding in Th9 cell treated with IFN-γ were analysed. Over-represented motifs shown as PWM (positional weight matrix) revealed by MEME/DREME analysis are shown. Associated piecharts indicate the frequency of designated motifs. ( f ) Non-polarized Th0 cells were transfected with either WT or the mutated M1 or M3 Il9 promoter-luciferase vectors (substitution mutation within the Il9 promoter IRF site is depicted above) together with empty vectors and/or IRF4. ( g ) ChIP-Seq analyses show IRF4 (upper lanes), IRF1 (middle lane) or H3K27-Ac (bottom two lanes) abundance. Below in grey boxes, potential IRF-binding sites are depicted including their distance from the Il9 transcription start site. ( h ) Normalized read counts from IRF4-ChIP-Seq in Th9 cells in the absence or presence of IFN-γ for CNS0, 1 and 2 are shown. ( a – c , f ) Data are combined from three independent experiments and show mean±s.d. * P

    Article Snippet: For intracellular staining of transcription factors after differentiation, cells were fixed with a Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Dilutent (eBioscience) and then stained with: anti-Foxp3-PE (FJK-16, eBioscience), anti-T-bet-PE (eBio4B10, eBioscience), anti-GATA-3-eF660 (TWAJ, eBioscience), anti-IRF4-AIFI647, (3E4, eBioscience) or anti-IRF1-PE (D5E4, Cell Signaling) mAbs.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation, Sequencing

    The IFN-γ/IRF1 pathway restricts Th9-dependent allergic airway inflammation. WT or Irf1 −/− Th9 cells treated with IFN-γ were adoptively transferred into Rag2 −/− mice, which were then challenged with nebulized OVA in the absence or presence of IL-9-neutralizing monoclonal antibodies (anti-IL-9) or control rat IgG (Ctrl) antibodies as outlined in Supplementary Fig. 6 . ( a , b , f , g ) Cell numbers in the BAL fluid are displayed. ( b , g ) Numbers of mucus-producing goblet cells evaluated by periodic acid-Schiff staining. ( c – e ) Lung cells were stimulated with 2 mM OVA 323–339 for 3 days. IL-9, IL-13 and IFN-γ production was determined in supernatants by ELISA. ( h ) Tissue inflammation was evaluated with haematoxylin and eosin staining, original magnification × 100. ( a – g ) Data are shown as the mean±s.e.m. of 10–15 mice combining two independent experiments. ( a – h ) The experiments were repeated four times with consistent results. * P

    Journal: Nature Communications

    Article Title: Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

    doi: 10.1038/ncomms15366

    Figure Lengend Snippet: The IFN-γ/IRF1 pathway restricts Th9-dependent allergic airway inflammation. WT or Irf1 −/− Th9 cells treated with IFN-γ were adoptively transferred into Rag2 −/− mice, which were then challenged with nebulized OVA in the absence or presence of IL-9-neutralizing monoclonal antibodies (anti-IL-9) or control rat IgG (Ctrl) antibodies as outlined in Supplementary Fig. 6 . ( a , b , f , g ) Cell numbers in the BAL fluid are displayed. ( b , g ) Numbers of mucus-producing goblet cells evaluated by periodic acid-Schiff staining. ( c – e ) Lung cells were stimulated with 2 mM OVA 323–339 for 3 days. IL-9, IL-13 and IFN-γ production was determined in supernatants by ELISA. ( h ) Tissue inflammation was evaluated with haematoxylin and eosin staining, original magnification × 100. ( a – g ) Data are shown as the mean±s.e.m. of 10–15 mice combining two independent experiments. ( a – h ) The experiments were repeated four times with consistent results. * P

    Article Snippet: For intracellular staining of transcription factors after differentiation, cells were fixed with a Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Dilutent (eBioscience) and then stained with: anti-Foxp3-PE (FJK-16, eBioscience), anti-T-bet-PE (eBio4B10, eBioscience), anti-GATA-3-eF660 (TWAJ, eBioscience), anti-IRF4-AIFI647, (3E4, eBioscience) or anti-IRF1-PE (D5E4, Cell Signaling) mAbs.

    Techniques: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

    The IFN-γ/IRF1 pathway upregulates IFN/Th1-associated genes in Th9 cells. ( a ) ChIP-Seq analyses of Th9 cells with or without IFN-γ treatment. Cells were crosslinked with formaldehyde and immunoprecipitated with anti-H3K27-Ac antibodies. Massive parallel sequencing of immunoprecipitated DNA was performed. Numbers in the Venn diagram represent H3K27-Ac peaks. ( b ) Naive CD4 + T cells isolated from WT and Irf1 −/− mice were cultured under Th9 conditions in the presence of IFN-γ. Total RNA was purified from the cells and RNA-Seq was performed (three independent biological samples). Heatmap shows colour-coded z -scored fragments per kilobase of exon per million fragments mapped ( P

    Journal: Nature Communications

    Article Title: Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

    doi: 10.1038/ncomms15366

    Figure Lengend Snippet: The IFN-γ/IRF1 pathway upregulates IFN/Th1-associated genes in Th9 cells. ( a ) ChIP-Seq analyses of Th9 cells with or without IFN-γ treatment. Cells were crosslinked with formaldehyde and immunoprecipitated with anti-H3K27-Ac antibodies. Massive parallel sequencing of immunoprecipitated DNA was performed. Numbers in the Venn diagram represent H3K27-Ac peaks. ( b ) Naive CD4 + T cells isolated from WT and Irf1 −/− mice were cultured under Th9 conditions in the presence of IFN-γ. Total RNA was purified from the cells and RNA-Seq was performed (three independent biological samples). Heatmap shows colour-coded z -scored fragments per kilobase of exon per million fragments mapped ( P

    Article Snippet: For intracellular staining of transcription factors after differentiation, cells were fixed with a Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Dilutent (eBioscience) and then stained with: anti-Foxp3-PE (FJK-16, eBioscience), anti-T-bet-PE (eBio4B10, eBioscience), anti-GATA-3-eF660 (TWAJ, eBioscience), anti-IRF4-AIFI647, (3E4, eBioscience) or anti-IRF1-PE (D5E4, Cell Signaling) mAbs.

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Sequencing, Isolation, Mouse Assay, Cell Culture, Purification, RNA Sequencing Assay

    IFN-γ/STAT1-induced IRF1 suppresses IL-9 production in human Th9 cells. Naive CD4 + CD25 − CD45RA + CD45RO − T cells were isolated from human PBMCs and cultured under Th0 or Th9 conditions in the presence or absence of human IFN-γ as indicated. ( a ) Intracellular staining for IL-9 and IFN-γ after 3 days of culture (bars to the right give mean±s.d. of three independent experiments). ( b ) Expression of IL9 and IFNG mRNA on day 2 in Th0 and Th9 cells; results were normalized to 18S and are presented relative to Th0 cells. ( c ) ELISA of IL-9 in supernatants of Th0 and Th9 cultures after 3 days. ( d ) Phospho-flow for p-STAT1 in Th9 cells with or without IFN-γ treatment, day 2 of culture (values show mean fluoresence intensity). ( e ) Intracellular staining for IRF1 and IRF4, day 2 of culture (bars to the right give mean±s.d. of three independent experiments). ( f – h ) Human naive CD4 + CD25 − CD45RA + CD45RO − T cells were activated under Th0 condition for 16 h, then transfected with scrambled (scr) or IRF1-specific siRNA (siIRF1) and then cultured for further 72 h under Th9 conditions with/without IFN-γ. ( f ) Intracellular staining of IRF1 in scr- or siIRF1-transfected cells 24 h post transfection upon culture under Th9 conditions with IFN-γ. ( g ) Expression of IL9 mRNA in transfected cells at 48 h post transfection; results were normalized to 18S and are presented relative to Th9 cells treated with scr siRNA and IFN-γ. ( h ) ELISA of IL-9 in supernatants of transfected cells 72 h post transfection. ( a – h ) Data are representative of three independent experiments with different donors; * P

    Journal: Nature Communications

    Article Title: Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

    doi: 10.1038/ncomms15366

    Figure Lengend Snippet: IFN-γ/STAT1-induced IRF1 suppresses IL-9 production in human Th9 cells. Naive CD4 + CD25 − CD45RA + CD45RO − T cells were isolated from human PBMCs and cultured under Th0 or Th9 conditions in the presence or absence of human IFN-γ as indicated. ( a ) Intracellular staining for IL-9 and IFN-γ after 3 days of culture (bars to the right give mean±s.d. of three independent experiments). ( b ) Expression of IL9 and IFNG mRNA on day 2 in Th0 and Th9 cells; results were normalized to 18S and are presented relative to Th0 cells. ( c ) ELISA of IL-9 in supernatants of Th0 and Th9 cultures after 3 days. ( d ) Phospho-flow for p-STAT1 in Th9 cells with or without IFN-γ treatment, day 2 of culture (values show mean fluoresence intensity). ( e ) Intracellular staining for IRF1 and IRF4, day 2 of culture (bars to the right give mean±s.d. of three independent experiments). ( f – h ) Human naive CD4 + CD25 − CD45RA + CD45RO − T cells were activated under Th0 condition for 16 h, then transfected with scrambled (scr) or IRF1-specific siRNA (siIRF1) and then cultured for further 72 h under Th9 conditions with/without IFN-γ. ( f ) Intracellular staining of IRF1 in scr- or siIRF1-transfected cells 24 h post transfection upon culture under Th9 conditions with IFN-γ. ( g ) Expression of IL9 mRNA in transfected cells at 48 h post transfection; results were normalized to 18S and are presented relative to Th9 cells treated with scr siRNA and IFN-γ. ( h ) ELISA of IL-9 in supernatants of transfected cells 72 h post transfection. ( a – h ) Data are representative of three independent experiments with different donors; * P

    Article Snippet: For intracellular staining of transcription factors after differentiation, cells were fixed with a Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Dilutent (eBioscience) and then stained with: anti-Foxp3-PE (FJK-16, eBioscience), anti-T-bet-PE (eBio4B10, eBioscience), anti-GATA-3-eF660 (TWAJ, eBioscience), anti-IRF4-AIFI647, (3E4, eBioscience) or anti-IRF1-PE (D5E4, Cell Signaling) mAbs.

    Techniques: Isolation, Cell Culture, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Transfection

    IRF1 limits IRF4-driven IL-9 production dose-dependently. ( a – c ) Naive CD44 − CD62L + CD4 + T cells were isolated from WT, Irf4 −/− , Irf1 −/− or Stat1 −/− mice and then treated under Th9 (TGF-β and IL-4) or Th0 (without skewing cytokines) conditions with/without IFN-γ as indicated. ( a , b ) After resting and reculture under Th9 conditions for 2 days, intracellular flow cytometric analysis for IRF1 and IRF4 were performed. Bars give geometric mean of fluorescence intensity (MFI). ( c ) Cells were restimulated and then IL-9 and IFN-γ production was detected by flow cytometry, bars to the right give mean of IL-9 + T cells. ( d , e ) Irf4 −/− CD4 + T cells were isolated, activated under Th0 condition overnight and then spin-infected with retroviruses as indicated: IRF4-GFP-RV, control-Thy1.1-RV, and IRF1-Thy1.1-RV. Thereafter, cells were cultured under Th9 conditions for 2 days, rested for 3 days and recultured under Th9 conditions for additional 2 days. Four subsets (I–IV) were selected for analysis of IL-9 production. Bars to the right show fold induction of IL-9 + T cells relative to GFP − Thy.1.1 − cells (subset I). ( e ) Six subsets (a through f) expressing increasing levels of GFP and Thy1.1 were selected for analysis of IL-9 production (left panel). Dot plot to the left is representative for four independent experiments. Graph to the right shows fold induction of IL-9 + T cells relative to GFP − Thy1.1 − cells (subset a) combined from four independent experiments. Histogram and contour-plots are representative of two ( a , b ), three ( c ) or four ( d , e ) independent experiments. Bars show mean±s.d. from combined two ( a , b ), three ( c ) or four ( d , e ) experiments. * P

    Journal: Nature Communications

    Article Title: Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

    doi: 10.1038/ncomms15366

    Figure Lengend Snippet: IRF1 limits IRF4-driven IL-9 production dose-dependently. ( a – c ) Naive CD44 − CD62L + CD4 + T cells were isolated from WT, Irf4 −/− , Irf1 −/− or Stat1 −/− mice and then treated under Th9 (TGF-β and IL-4) or Th0 (without skewing cytokines) conditions with/without IFN-γ as indicated. ( a , b ) After resting and reculture under Th9 conditions for 2 days, intracellular flow cytometric analysis for IRF1 and IRF4 were performed. Bars give geometric mean of fluorescence intensity (MFI). ( c ) Cells were restimulated and then IL-9 and IFN-γ production was detected by flow cytometry, bars to the right give mean of IL-9 + T cells. ( d , e ) Irf4 −/− CD4 + T cells were isolated, activated under Th0 condition overnight and then spin-infected with retroviruses as indicated: IRF4-GFP-RV, control-Thy1.1-RV, and IRF1-Thy1.1-RV. Thereafter, cells were cultured under Th9 conditions for 2 days, rested for 3 days and recultured under Th9 conditions for additional 2 days. Four subsets (I–IV) were selected for analysis of IL-9 production. Bars to the right show fold induction of IL-9 + T cells relative to GFP − Thy.1.1 − cells (subset I). ( e ) Six subsets (a through f) expressing increasing levels of GFP and Thy1.1 were selected for analysis of IL-9 production (left panel). Dot plot to the left is representative for four independent experiments. Graph to the right shows fold induction of IL-9 + T cells relative to GFP − Thy1.1 − cells (subset a) combined from four independent experiments. Histogram and contour-plots are representative of two ( a , b ), three ( c ) or four ( d , e ) independent experiments. Bars show mean±s.d. from combined two ( a , b ), three ( c ) or four ( d , e ) experiments. * P

    Article Snippet: For intracellular staining of transcription factors after differentiation, cells were fixed with a Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Dilutent (eBioscience) and then stained with: anti-Foxp3-PE (FJK-16, eBioscience), anti-T-bet-PE (eBio4B10, eBioscience), anti-GATA-3-eF660 (TWAJ, eBioscience), anti-IRF4-AIFI647, (3E4, eBioscience) or anti-IRF1-PE (D5E4, Cell Signaling) mAbs.

    Techniques: Isolation, Mouse Assay, Flow Cytometry, Fluorescence, Cytometry, Infection, Cell Culture, Expressing

    Complement opsonization of HIV-1 suppressed IRF1 and IRF7 but activated IRF3 signaling. DCs (1 × 10 6 /ml) were exposed to F-HIV, C-HIV, and CI-HIV at an MOI of 8 or were mock treated. mRNA and protein expression of IRF1 ( A and B ), IRF3 ( C and

    Journal: The Journal of Immunology Author Choice

    Article Title: Complement Opsonization of HIV-1 Results in Decreased Antiviral and Inflammatory Responses in Immature Dendritic Cells via CR3

    doi: 10.4049/jimmunol.1401781

    Figure Lengend Snippet: Complement opsonization of HIV-1 suppressed IRF1 and IRF7 but activated IRF3 signaling. DCs (1 × 10 6 /ml) were exposed to F-HIV, C-HIV, and CI-HIV at an MOI of 8 or were mock treated. mRNA and protein expression of IRF1 ( A and B ), IRF3 ( C and

    Article Snippet: iDCs (1 × 106 cells/ml) were exposed to F-HIV, C-HIV, or CI-HIV or mock for 0.5, 1, 3, or 6 h. For nuclear translocation the cells were fixed, permeabilized, stained with rabbit anti–NF-κB p65 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-IRF1 (Cell Signaling Technology, Danvers, MA), or mouse anti-IRF3 (Invitrogen, Stockholm, Sweden) Abs followed by Alexa Fluor 488–conjugated anti-rabbit (Abcam, Cambridge, U.K.) or Rhodamine Red-X–conjugated anti-mouse (Molecular Probes, Eugene, OR) secondary Abs and fluorescence mounting medium containing DAPI (Vector Laboratories, Peterborough, U.K.).

    Techniques: Expressing

    ISRE induction by ChX710 is dependent on MAVS and IRF1. ( a ) MAVS was silenced by siRNA transfection in ISRE-luciferase reporter cells. After 48 hours of cultures, cells were stimulated with ChX710. After 24 hours of incubation, luciferase induction was determined. ( b ) Same experiment as ( a ) using STING-specific siRNA. ( c ) Same experiment as ( a ) using IRF1-specific siRNA. ( d ) Same experiment as ( a ) using IRF3-specific siRNA. ( e,f ) IRF3 phosphorylation was determined by western-blot in HEK-293T ( e ) or A549 cells ( f ) treated for 24 hours with DMSO alone or ChX710 at 25 μM or 50 μM. ( g ) Schematic model of the signaling pathways induced by ChX710. IRF3 phosphorylation is induced, but this is insufficient to activate IFN-β expression. Data represent means ± SD of three independent experiments, except for ( e ) and ( f ) that corresponds to representative results. ** P

    Journal: Scientific Reports

    Article Title: Identification of a small molecule that primes the type I interferon response to cytosolic DNA

    doi: 10.1038/s41598-017-02776-z

    Figure Lengend Snippet: ISRE induction by ChX710 is dependent on MAVS and IRF1. ( a ) MAVS was silenced by siRNA transfection in ISRE-luciferase reporter cells. After 48 hours of cultures, cells were stimulated with ChX710. After 24 hours of incubation, luciferase induction was determined. ( b ) Same experiment as ( a ) using STING-specific siRNA. ( c ) Same experiment as ( a ) using IRF1-specific siRNA. ( d ) Same experiment as ( a ) using IRF3-specific siRNA. ( e,f ) IRF3 phosphorylation was determined by western-blot in HEK-293T ( e ) or A549 cells ( f ) treated for 24 hours with DMSO alone or ChX710 at 25 μM or 50 μM. ( g ) Schematic model of the signaling pathways induced by ChX710. IRF3 phosphorylation is induced, but this is insufficient to activate IFN-β expression. Data represent means ± SD of three independent experiments, except for ( e ) and ( f ) that corresponds to representative results. ** P

    Article Snippet: Proteins were detected using standard immunoblotting techniques using the following primary antibodies: anti-IRF1 rabbit monoclonal antibody was from Cell Signaling (Clone D5E4; Ref. 8478S), anti-IRF3 rabbit monoclonal antibody was from Cell Signaling (Clone D6I4C; Ref. 11904), anti-P-IRF3 (Ser386) rabbit polyclonal antibody was from Millipore (ABE501), anti-STAT1 mouse monoclonal antibody was from Cell Signaling (Clone 9H2; Ref. 9176), anti-STAT2 rabbit polyclonal antibody was from Cell Signaling (Ref. 4594), anti-MAVS rabbit polyclonal antibody was from Alexis (Ref AT107; 210–929-C100), anti-STING rabbit monoclonal antibody was from Cell Signaling (Clone D2P2F; Ref 13647), anti-ULK1 rabbit monoclonal antibody was from Cell Signaling (Clone D8H5; Ref 8054), and anti-β-Actin mouse monoclonal antibody was from Sigma-Aldrich (Clone AC-15; Ref. A5441).

    Techniques: Transfection, Luciferase, Incubation, Western Blot, Expressing