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    Addgene inc pmig irf4
    (A) In silico analysis of <t>Irf4</t> and Slc2a3 genes in CD8+ T cells for chromatin accessibility measured by DNase I hypersensitivity analysis (Bevington et al., 2016) and ATAC sequencing (Mognol et al., 2017) and binding of NFATc1 (Klein-Hessling, 2017) and NFATc2 (Martinez et al. 2015). (B) Luciferase reporter analyses of the Slc2a3 promoter (Prom.) with or without the –16 kb regulatory element in HEK293T cells. Cells were stimulated with PMA and ionomycin in the presence or absence of CsA; means ± SEM of 6–11 independent transfections. (C and D) Analysis of Irf4, Hif1a, Slc2a3 and Hk2 gene expression by qRT-PCR in CD4+ T cells from (C) WT and Nfatc1fl/flNfatc2−/−Cd4cre mice and (D) WT and Rosa26LSL-caNfatc1 dLckcre (caNfatc1KI) mice. Data represent the means ± SEM of 3–4 mice. (E and F) Analysis of (E) GLUT3 expression by flow cytometry and (F) expansion of primary mouse CD4+ T cells after deletion of Slc2a3 (GLUT3) with small guide (sg) RNAs. Shown in (F) are the ratios of T cells transduced with control or Slc2a3-specific sgRNAs compared to non-transduced T cells following αCD3 and αCD28 stimulation; means ± SEM of 4 mice. (G–I) Rescue of (G) IRF4 expression, (H) GLUT1 expression and (I) 2-NBDG uptake by retroviral transduction of WT and Stim1fl/flStim2fl/flCd4cre (Stim1/2CD4) CD4+ T cells with caNFATc1 or empty control vector; means ± SEM of 3–4 mice. (J–L) Partial rescue of the clonal expansion of adoptively transferred LCMV-specific, SOCE-deficient T cells by retroviral expression of caNFATc1 or GLUT1. (J) Experimental design, (K) flow cytometric analyses of the frequencies and (L) absolute numbers of caNFATc1 or GLUT1 transduced CD4+ T cells from WT and Stim1/2CD4 SMARTA mice 8 d after LCMV infection; means ± SEM of 7–8 host mice from 3 independent experiments. See also Figure S5.
    Pmig Irf4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) In silico analysis of Irf4 and Slc2a3 genes in CD8+ T cells for chromatin accessibility measured by DNase I hypersensitivity analysis (Bevington et al., 2016) and ATAC sequencing (Mognol et al., 2017) and binding of NFATc1 (Klein-Hessling, 2017) and NFATc2 (Martinez et al. 2015). (B) Luciferase reporter analyses of the Slc2a3 promoter (Prom.) with or without the –16 kb regulatory element in HEK293T cells. Cells were stimulated with PMA and ionomycin in the presence or absence of CsA; means ± SEM of 6–11 independent transfections. (C and D) Analysis of Irf4, Hif1a, Slc2a3 and Hk2 gene expression by qRT-PCR in CD4+ T cells from (C) WT and Nfatc1fl/flNfatc2−/−Cd4cre mice and (D) WT and Rosa26LSL-caNfatc1 dLckcre (caNfatc1KI) mice. Data represent the means ± SEM of 3–4 mice. (E and F) Analysis of (E) GLUT3 expression by flow cytometry and (F) expansion of primary mouse CD4+ T cells after deletion of Slc2a3 (GLUT3) with small guide (sg) RNAs. Shown in (F) are the ratios of T cells transduced with control or Slc2a3-specific sgRNAs compared to non-transduced T cells following αCD3 and αCD28 stimulation; means ± SEM of 4 mice. (G–I) Rescue of (G) IRF4 expression, (H) GLUT1 expression and (I) 2-NBDG uptake by retroviral transduction of WT and Stim1fl/flStim2fl/flCd4cre (Stim1/2CD4) CD4+ T cells with caNFATc1 or empty control vector; means ± SEM of 3–4 mice. (J–L) Partial rescue of the clonal expansion of adoptively transferred LCMV-specific, SOCE-deficient T cells by retroviral expression of caNFATc1 or GLUT1. (J) Experimental design, (K) flow cytometric analyses of the frequencies and (L) absolute numbers of caNFATc1 or GLUT1 transduced CD4+ T cells from WT and Stim1/2CD4 SMARTA mice 8 d after LCMV infection; means ± SEM of 7–8 host mice from 3 independent experiments. See also Figure S5.

    Journal: Immunity

    Article Title: Store-operated Ca 2+ entry controls clonal expansion of T cells through metabolic reprogramming

    doi: 10.1016/j.immuni.2017.09.003

    Figure Lengend Snippet: (A) In silico analysis of Irf4 and Slc2a3 genes in CD8+ T cells for chromatin accessibility measured by DNase I hypersensitivity analysis (Bevington et al., 2016) and ATAC sequencing (Mognol et al., 2017) and binding of NFATc1 (Klein-Hessling, 2017) and NFATc2 (Martinez et al. 2015). (B) Luciferase reporter analyses of the Slc2a3 promoter (Prom.) with or without the –16 kb regulatory element in HEK293T cells. Cells were stimulated with PMA and ionomycin in the presence or absence of CsA; means ± SEM of 6–11 independent transfections. (C and D) Analysis of Irf4, Hif1a, Slc2a3 and Hk2 gene expression by qRT-PCR in CD4+ T cells from (C) WT and Nfatc1fl/flNfatc2−/−Cd4cre mice and (D) WT and Rosa26LSL-caNfatc1 dLckcre (caNfatc1KI) mice. Data represent the means ± SEM of 3–4 mice. (E and F) Analysis of (E) GLUT3 expression by flow cytometry and (F) expansion of primary mouse CD4+ T cells after deletion of Slc2a3 (GLUT3) with small guide (sg) RNAs. Shown in (F) are the ratios of T cells transduced with control or Slc2a3-specific sgRNAs compared to non-transduced T cells following αCD3 and αCD28 stimulation; means ± SEM of 4 mice. (G–I) Rescue of (G) IRF4 expression, (H) GLUT1 expression and (I) 2-NBDG uptake by retroviral transduction of WT and Stim1fl/flStim2fl/flCd4cre (Stim1/2CD4) CD4+ T cells with caNFATc1 or empty control vector; means ± SEM of 3–4 mice. (J–L) Partial rescue of the clonal expansion of adoptively transferred LCMV-specific, SOCE-deficient T cells by retroviral expression of caNFATc1 or GLUT1. (J) Experimental design, (K) flow cytometric analyses of the frequencies and (L) absolute numbers of caNFATc1 or GLUT1 transduced CD4+ T cells from WT and Stim1/2CD4 SMARTA mice 8 d after LCMV infection; means ± SEM of 7–8 host mice from 3 independent experiments. See also Figure S5.

    Article Snippet: pMIG-IRF4 , Addgene , Cat# 58987.

    Techniques: In Silico, Sequencing, Binding Assay, Luciferase, Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Transduction, Plasmid Preparation, Infection

    (A) Analysis of SOCE following thapsigargin (TG) stimulation in CD4+ T cells from a healthy donor (Mother) and a patient homozygous for a null mutation in STIM1 (p.L374P). (B) CFSE dilution of healthy donor and STIM1 p.L374P patient CD4+ T cells 3 d after αCD3 and αCD28 stimulation in the presence or absence of 1 μM FK506. (C–E) Analysis of (C) cell size (FSC-A), (D) GLUT1 protein expression and (E) uptake of 2-NBDG by flow cytometry. CD4+ T cells from the healthy donor and STIM1 p.L374P patient were stimulated for 24 h with αCD3 and αCD28 in the presence or absence of 1 μM FK506. (F and G) Analysis of (F) SLC2A1, HK2 and PGK1 and (G) MYC, IRF4 and PPRC1 gene expression in CD4+ T cells of a healthy donor and the STIM1 p.L374P patient stimulated as in (B). See also Figure S6.

    Journal: Immunity

    Article Title: Store-operated Ca 2+ entry controls clonal expansion of T cells through metabolic reprogramming

    doi: 10.1016/j.immuni.2017.09.003

    Figure Lengend Snippet: (A) Analysis of SOCE following thapsigargin (TG) stimulation in CD4+ T cells from a healthy donor (Mother) and a patient homozygous for a null mutation in STIM1 (p.L374P). (B) CFSE dilution of healthy donor and STIM1 p.L374P patient CD4+ T cells 3 d after αCD3 and αCD28 stimulation in the presence or absence of 1 μM FK506. (C–E) Analysis of (C) cell size (FSC-A), (D) GLUT1 protein expression and (E) uptake of 2-NBDG by flow cytometry. CD4+ T cells from the healthy donor and STIM1 p.L374P patient were stimulated for 24 h with αCD3 and αCD28 in the presence or absence of 1 μM FK506. (F and G) Analysis of (F) SLC2A1, HK2 and PGK1 and (G) MYC, IRF4 and PPRC1 gene expression in CD4+ T cells of a healthy donor and the STIM1 p.L374P patient stimulated as in (B). See also Figure S6.

    Article Snippet: pMIG-IRF4 , Addgene , Cat# 58987.

    Techniques: Mutagenesis, Expressing, Flow Cytometry

    KEY RESOURCES TABLE

    Journal: Immunity

    Article Title: Store-operated Ca 2+ entry controls clonal expansion of T cells through metabolic reprogramming

    doi: 10.1016/j.immuni.2017.09.003

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: pMIG-IRF4 , Addgene , Cat# 58987.

    Techniques: Recombinant, Transfection, Staining, DNA Extraction, SYBR Green Assay, Sequencing, Plasmid Preparation, Software, Mass Spectrometry, Microscopy, Flow Cytometry