Review



rabbit ant human slc19a1 pab  (Boster Bio)


Bioz Verified Symbol Boster Bio is a verified supplier
Bioz Manufacturer Symbol Boster Bio manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 90
      Buy from Supplier

    Structured Review

    Boster Bio rabbit ant human slc19a1 pab
    <t>SLC19A1</t> is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 μg/ml) or 2’3’-cGAMP (15 μg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (10 μg/ml), or 3’3’-CDA (20 μg/ml). After 18-22h, tdTomato expression was quantified as in (a). c, Induction of IFNB mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 μg/ml 2’3’-RR CDA. d, Control THP-1 cells and SLC19A1–1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (15 μg/ml), or hIFN-β (100 ng/ml) and analyzed as in (a). e, Control THP-1 cells (n=7 clonal lines) and SLC19A1 −/− cells ( 19A1 −/− ; n=9 clonal lines) were exposed to 2’3’-RR CDA (2.22 μg/ml), 2’3’-cGAMP (10 μg/ml), and tdTomato reporter expression was analyzed as in (a). Mean ± SEM are shown. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated and analyzed as in (b). g, THP-1 cells were incubated with increasing concentrations of the competitive inhibitors methotrexate, 5-methyl tetrahydrofolate (5-me-THF) or DMSO as vehicle control, before stimulating with 2’3’-RR CDA (1.25 μg/ml), 2’3’-cGAMP (15 μg/ml) or hIFN-β (100 ng/ml). Cells were analyzed as in (a). For each stimulant, the data were normalized to the DMSO controls. In panels b-d and f-g, mean ± SEM of n=3 biological replicates are shown. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test for the comparison to stimulated control cells (b-d), unpaired two-tailed Student’s t tests for (e), or two-way ANOVA followed by uncorrected Fisher’s LSD tests (f). *a P = 0.0002; *b P = 0.0013; *c P = 0.0005; *d P =0.0006; **** P ≤ 0.0001; n.s. not significant.
    Rabbit Ant Human Slc19a1 Pab, supplied by Boster Bio, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+ant+human+slc19a1+pab/pmc06785039-22-26-29?v=Boster+Bio
    Average 90 stars, based on 3 article reviews
    rabbit ant human slc19a1 pab - by Bioz Stars, 2026-08
    90/100 stars

    Images

    1) Product Images from "SLC19A1 transports immunoreactive cyclic dinucleotides"

    Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

    Journal: Nature

    doi: 10.1038/s41586-019-1553-0

    SLC19A1 is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 μg/ml) or 2’3’-cGAMP (15 μg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (10 μg/ml), or 3’3’-CDA (20 μg/ml). After 18-22h, tdTomato expression was quantified as in (a). c, Induction of IFNB mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 μg/ml 2’3’-RR CDA. d, Control THP-1 cells and SLC19A1–1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (15 μg/ml), or hIFN-β (100 ng/ml) and analyzed as in (a). e, Control THP-1 cells (n=7 clonal lines) and SLC19A1 −/− cells ( 19A1 −/− ; n=9 clonal lines) were exposed to 2’3’-RR CDA (2.22 μg/ml), 2’3’-cGAMP (10 μg/ml), and tdTomato reporter expression was analyzed as in (a). Mean ± SEM are shown. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated and analyzed as in (b). g, THP-1 cells were incubated with increasing concentrations of the competitive inhibitors methotrexate, 5-methyl tetrahydrofolate (5-me-THF) or DMSO as vehicle control, before stimulating with 2’3’-RR CDA (1.25 μg/ml), 2’3’-cGAMP (15 μg/ml) or hIFN-β (100 ng/ml). Cells were analyzed as in (a). For each stimulant, the data were normalized to the DMSO controls. In panels b-d and f-g, mean ± SEM of n=3 biological replicates are shown. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test for the comparison to stimulated control cells (b-d), unpaired two-tailed Student’s t tests for (e), or two-way ANOVA followed by uncorrected Fisher’s LSD tests (f). *a P = 0.0002; *b P = 0.0013; *c P = 0.0005; *d P =0.0006; **** P ≤ 0.0001; n.s. not significant.
    Figure Legend Snippet: SLC19A1 is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 μg/ml) or 2’3’-cGAMP (15 μg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (10 μg/ml), or 3’3’-CDA (20 μg/ml). After 18-22h, tdTomato expression was quantified as in (a). c, Induction of IFNB mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 μg/ml 2’3’-RR CDA. d, Control THP-1 cells and SLC19A1–1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (15 μg/ml), or hIFN-β (100 ng/ml) and analyzed as in (a). e, Control THP-1 cells (n=7 clonal lines) and SLC19A1 −/− cells ( 19A1 −/− ; n=9 clonal lines) were exposed to 2’3’-RR CDA (2.22 μg/ml), 2’3’-cGAMP (10 μg/ml), and tdTomato reporter expression was analyzed as in (a). Mean ± SEM are shown. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated and analyzed as in (b). g, THP-1 cells were incubated with increasing concentrations of the competitive inhibitors methotrexate, 5-methyl tetrahydrofolate (5-me-THF) or DMSO as vehicle control, before stimulating with 2’3’-RR CDA (1.25 μg/ml), 2’3’-cGAMP (15 μg/ml) or hIFN-β (100 ng/ml). Cells were analyzed as in (a). For each stimulant, the data were normalized to the DMSO controls. In panels b-d and f-g, mean ± SEM of n=3 biological replicates are shown. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test for the comparison to stimulated control cells (b-d), unpaired two-tailed Student’s t tests for (e), or two-way ANOVA followed by uncorrected Fisher’s LSD tests (f). *a P = 0.0002; *b P = 0.0013; *c P = 0.0005; *d P =0.0006; **** P ≤ 0.0001; n.s. not significant.

    Techniques Used: Expressing, Transduction, Control, Flow Cytometry, Plasmid Preparation, Incubation, Comparison, Two Tailed Test

    a , Thin layer chromatography (TLC) analysis of [ 32 P] ATP standard (STD) and enzymatically synthesized [ 32 P] 2’3’-cGAMP. 2’3’-cGAMP was purified on STING resin. Unbound nucleotides flowed through the resin (STING FT). Following four washes, the bound [32P] 2’3’-cGAMP was eluted over three fractions. b , DRaCALA binding analysis of [ 32 P] 2’3’-cGAMP to STING C-terminal domain (CTD) in the presence of competing unlabeled nucleotides (200 μM). c , Thin layer chromatography (TLC) analysis of [ 32 P]-ATP and enzymatically synthesized [ 32 P]-2’3’-cGAMP and [ 32 P]-c-di-AMP. d , Binding titration of [ 32 P] 2’3’-cGAMP or [ 32 P] c-di-AMP to mSTING C-Terminal Domain (CTD), determined with DRaCALA assays. Red dashed lines represent the 95% confidence interval for the non-linear regression. e , Time course of [ 32 P] 2’3’-cGAMP (left panel) or [ 32 P] 3’3’-CDA (right panel) uptake in THP-1 monocytes. f , TLC analysis (left panel) and STING-binding (DRaCALA) (right panel) of [ 32 P] ATP standard, or [ 32 P] 2’3’-cGAMP recovered from supernatants of THP-1 monocytes at the indicated time points. g , h , Effect of cell culture medium pH on [ 32 P] 2’3’-cGAMP uptake in THP-1 (g) monocytes or U937 monocytes (h). i , Time course of [ 32 P] 2’3’-cGAMP uptake by CIR cells transduced (tr.) with empty vector or SLC19A1 expression vector. j, mRNA expression levels of SLC19A1 (SLC.) in K562 cells expressing control shRNAs (sh1 and sh2) or an SLC19A1 -targeting shRNA (sh9). k, mRNA expression levels of CXCL10 in K562 cells described in panel j, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. l, [ 3 H]-Methotrexate uptake in K562 cells described in panel j, 1h after exposure to [ 3 H]-Methotrexate. m , Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells described in panel j. n , Time course of [ 32 P] 2’3’-cGAMP uptake in U937 monocytes in the presence or absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. o . Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells in the presence or absence of 500 μM competing, unlabeled (anti-) folates or sulfasalazine. p , Competition uptake assay of [ 32 P] 2’3’-cGAMP uptake in THP-1 cells in the presence of varying concentrations of competing, unlabeled 5-me-THF (IC50 = 4.10 ± 0.16 nM), methotrexate (IC50 = 54.83 ± 5.08 nM), 2’3’-cGAMP (IC50 = 1.89 ± 0.11 μM), sulfasalazine ((IC50 = 2.06 ± 0.17 μM), and folic acid (IC50 = 4.79 ± 0.08 μM). q , Trans-stimulation of [ 32 P] 2’3’-cGAMP influx in THP-1 cells by 5-me-THF. Cells were preloaded with indicated concentrations of 5-me-THF for 30 min. Cells were washed and incubated with [ 32 P] 2’3’-cGAMP for one hour. r , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated human PBMCs from four healthy donors. In panel a and c, data are representative of three independent experiments with similar results. In panel b, data are means of n=2 technical replicates and are representative of three independent experiments. In panel d and f, data are means of n=2 technical replicates and are representative of two independent experiments. In panel e and m, data are means ± SD of n=3 technical replicates and are representative of three independent experiments. In panel g, h, i, and n-r, data are means ± n=3 technical replicates and are representative of two independent experiments. In panel j and k, data are means ± SEM of n=3 biologically independent experiments. In panel l, data are means of n=2 biologically independent experiments.
    Figure Legend Snippet: a , Thin layer chromatography (TLC) analysis of [ 32 P] ATP standard (STD) and enzymatically synthesized [ 32 P] 2’3’-cGAMP. 2’3’-cGAMP was purified on STING resin. Unbound nucleotides flowed through the resin (STING FT). Following four washes, the bound [32P] 2’3’-cGAMP was eluted over three fractions. b , DRaCALA binding analysis of [ 32 P] 2’3’-cGAMP to STING C-terminal domain (CTD) in the presence of competing unlabeled nucleotides (200 μM). c , Thin layer chromatography (TLC) analysis of [ 32 P]-ATP and enzymatically synthesized [ 32 P]-2’3’-cGAMP and [ 32 P]-c-di-AMP. d , Binding titration of [ 32 P] 2’3’-cGAMP or [ 32 P] c-di-AMP to mSTING C-Terminal Domain (CTD), determined with DRaCALA assays. Red dashed lines represent the 95% confidence interval for the non-linear regression. e , Time course of [ 32 P] 2’3’-cGAMP (left panel) or [ 32 P] 3’3’-CDA (right panel) uptake in THP-1 monocytes. f , TLC analysis (left panel) and STING-binding (DRaCALA) (right panel) of [ 32 P] ATP standard, or [ 32 P] 2’3’-cGAMP recovered from supernatants of THP-1 monocytes at the indicated time points. g , h , Effect of cell culture medium pH on [ 32 P] 2’3’-cGAMP uptake in THP-1 (g) monocytes or U937 monocytes (h). i , Time course of [ 32 P] 2’3’-cGAMP uptake by CIR cells transduced (tr.) with empty vector or SLC19A1 expression vector. j, mRNA expression levels of SLC19A1 (SLC.) in K562 cells expressing control shRNAs (sh1 and sh2) or an SLC19A1 -targeting shRNA (sh9). k, mRNA expression levels of CXCL10 in K562 cells described in panel j, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. l, [ 3 H]-Methotrexate uptake in K562 cells described in panel j, 1h after exposure to [ 3 H]-Methotrexate. m , Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells described in panel j. n , Time course of [ 32 P] 2’3’-cGAMP uptake in U937 monocytes in the presence or absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. o . Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells in the presence or absence of 500 μM competing, unlabeled (anti-) folates or sulfasalazine. p , Competition uptake assay of [ 32 P] 2’3’-cGAMP uptake in THP-1 cells in the presence of varying concentrations of competing, unlabeled 5-me-THF (IC50 = 4.10 ± 0.16 nM), methotrexate (IC50 = 54.83 ± 5.08 nM), 2’3’-cGAMP (IC50 = 1.89 ± 0.11 μM), sulfasalazine ((IC50 = 2.06 ± 0.17 μM), and folic acid (IC50 = 4.79 ± 0.08 μM). q , Trans-stimulation of [ 32 P] 2’3’-cGAMP influx in THP-1 cells by 5-me-THF. Cells were preloaded with indicated concentrations of 5-me-THF for 30 min. Cells were washed and incubated with [ 32 P] 2’3’-cGAMP for one hour. r , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated human PBMCs from four healthy donors. In panel a and c, data are representative of three independent experiments with similar results. In panel b, data are means of n=2 technical replicates and are representative of three independent experiments. In panel d and f, data are means of n=2 technical replicates and are representative of two independent experiments. In panel e and m, data are means ± SD of n=3 technical replicates and are representative of three independent experiments. In panel g, h, i, and n-r, data are means ± n=3 technical replicates and are representative of two independent experiments. In panel j and k, data are means ± SEM of n=3 biologically independent experiments. In panel l, data are means of n=2 biologically independent experiments.

    Techniques Used: Thin Layer Chromatography, Synthesized, Purification, Binding Assay, Titration, Cell Culture, Plasmid Preparation, Expressing, Control, shRNA, Incubation

    a, mRNA expression levels of Slc19a1 in mouse L1210 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh4 and sh6). b, mRNA expression levels of Cxcl10 in L1210 cells described in panel a stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. c, [ 3 H]-Methotrexate uptake in L1210 cells described in panel a 1h after exposure to [ 3 H]-Methotrexate. d, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells described in panel a. e, mRNA expression levels of Slc19a1 in mouse C1498 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh6). f, mRNA expression levels of Cxcl10 in the C1498 cells described in panel e, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. g, [ 3 H]-Methotrexate uptake in C1498 cells described in panel e 1h after exposure to [ 3 H]-Methotrexate. h , Time course of [ 32 P] 2’3’-cGAMP uptake in C1498 cells transduced with a non-targeting control shRNA (control) or Slc19a1 shRNA. i, j, mRNA expression levels of Slc19a1 in mouse bone marrow-derived macrophages (BMM) (i) or mouse bone marrow-derived dendritic cells (BMDCs) (j) transduced or not with control shRNAs (sh1 and 2) or an shRNA targeting Slc19a1 . k, l, mRNA expression of the Cxcl10 in cells described in panels i and j stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. m, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. n, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes pretreated or not with NHS-methotrexate (MTX) (5 μM). o, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells pretreated or not with NHS-MTX (5 μM). In panels a-c, e-g, j and l, data are means of n=2 biologically independent experiments. In panel d, h, i, k, n and o, data are means ± SD of n=3 technical replicates and are representative of two independent experiments. In panel m, data are means of n=2 technical replicates and are representative of two independent experiments. In time course experiments (d, h, m, n, o), data are presented as counts per minute (cpm) normalized to cell count.
    Figure Legend Snippet: a, mRNA expression levels of Slc19a1 in mouse L1210 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh4 and sh6). b, mRNA expression levels of Cxcl10 in L1210 cells described in panel a stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. c, [ 3 H]-Methotrexate uptake in L1210 cells described in panel a 1h after exposure to [ 3 H]-Methotrexate. d, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells described in panel a. e, mRNA expression levels of Slc19a1 in mouse C1498 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh6). f, mRNA expression levels of Cxcl10 in the C1498 cells described in panel e, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. g, [ 3 H]-Methotrexate uptake in C1498 cells described in panel e 1h after exposure to [ 3 H]-Methotrexate. h , Time course of [ 32 P] 2’3’-cGAMP uptake in C1498 cells transduced with a non-targeting control shRNA (control) or Slc19a1 shRNA. i, j, mRNA expression levels of Slc19a1 in mouse bone marrow-derived macrophages (BMM) (i) or mouse bone marrow-derived dendritic cells (BMDCs) (j) transduced or not with control shRNAs (sh1 and 2) or an shRNA targeting Slc19a1 . k, l, mRNA expression of the Cxcl10 in cells described in panels i and j stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. m, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. n, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes pretreated or not with NHS-methotrexate (MTX) (5 μM). o, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells pretreated or not with NHS-MTX (5 μM). In panels a-c, e-g, j and l, data are means of n=2 biologically independent experiments. In panel d, h, i, k, n and o, data are means ± SD of n=3 technical replicates and are representative of two independent experiments. In panel m, data are means of n=2 technical replicates and are representative of two independent experiments. In time course experiments (d, h, m, n, o), data are presented as counts per minute (cpm) normalized to cell count.

    Techniques Used: Expressing, Control, shRNA, Transduction, Derivative Assay, Cell Counting

    SLC19A1 transports CDNs. a , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control CRISPRi gRNA or SLC19A1 CRISPRi gRNA (right panel). b , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by K562 cells transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control shRNA (control) or SLC19A1 shRNA (right panel). c , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated THP-1 (left panel) and K562 (right panel) cells. d , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes in the presence and absence of 100 μM competing, unlabeled cyclic dinucleotides (left panel) or 200 μM competing, unlabeled nucleotides (right panel). e , Time course of [ 32 P] 2’3’-cGAMP uptake in THP-1 monocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. f , Normalized [ 32 P] 2’3’-cGAMP uptake after three hours in human PBMCs from six healthy donors in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. g , Coomassie staining and western blot analysis of pulldowns by 2’3’-cGAMP (+) or control (−) Sepharose of mSTING-CTD or hSLC19A1. h , Western blot analysis of hSLC19A1 affinity purification (AP) with 2’3’-cGAMP Sepharose in the absence (−) or presence (+) of free, unbound 2’3’-cGAMP, 5-me-THF, or methotrexate (250 μM). In panels a-d, data are means ± SEM of n=3 biological replicates. In panel e, data are means ± SD of n=3 technical replicates and data are representative of three independent experiments. In panel f, data are means ± SD of n=6 healthy donors conducted over two independent experiments. Panels g and h are representative of two independent experiments; for gel source data, see . Statistical analyses were performed using unpaired, two-tailed Student’s t-tests (a-d), or a one-way ANOVA followed by a Tukey’s post-test (f). **** P ≤ 0.0001.
    Figure Legend Snippet: SLC19A1 transports CDNs. a , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control CRISPRi gRNA or SLC19A1 CRISPRi gRNA (right panel). b , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by K562 cells transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control shRNA (control) or SLC19A1 shRNA (right panel). c , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated THP-1 (left panel) and K562 (right panel) cells. d , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes in the presence and absence of 100 μM competing, unlabeled cyclic dinucleotides (left panel) or 200 μM competing, unlabeled nucleotides (right panel). e , Time course of [ 32 P] 2’3’-cGAMP uptake in THP-1 monocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. f , Normalized [ 32 P] 2’3’-cGAMP uptake after three hours in human PBMCs from six healthy donors in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. g , Coomassie staining and western blot analysis of pulldowns by 2’3’-cGAMP (+) or control (−) Sepharose of mSTING-CTD or hSLC19A1. h , Western blot analysis of hSLC19A1 affinity purification (AP) with 2’3’-cGAMP Sepharose in the absence (−) or presence (+) of free, unbound 2’3’-cGAMP, 5-me-THF, or methotrexate (250 μM). In panels a-d, data are means ± SEM of n=3 biological replicates. In panel e, data are means ± SD of n=3 technical replicates and data are representative of three independent experiments. In panel f, data are means ± SD of n=6 healthy donors conducted over two independent experiments. Panels g and h are representative of two independent experiments; for gel source data, see . Statistical analyses were performed using unpaired, two-tailed Student’s t-tests (a-d), or a one-way ANOVA followed by a Tukey’s post-test (f). **** P ≤ 0.0001.

    Techniques Used: Transduction, Plasmid Preparation, Control, Expressing, shRNA, Staining, Western Blot, Affinity Purification, Two Tailed Test

    a , Sodium dodecyl sulfate (SDS)-PAGE analysis followed by Coomassie blue staining of His-tagged human SLC19A1 (Ni-NTA affinity-purified) pull-downs with Sepharose beads coupled with 2’3’-cGAMP (+) or control Sepharose beads (−). Input is shown in the right panel. b , Western blots of the samples in a with anti-SLC19A1 antibody. c , Pull-downs of SLC19A1 competed with CDNs. His-tagged SLC19A1 was incubated with no CDN or with the indicated competing CDNs (250 μM) before pulldowns with 2’3’-cGAMP-Sepharose, followed by SDS-PAGE and Western blotting with an anti-SLC19A1 antibody. A pulldown with control Sepharose is shown for comparison. For gel source data, see . d , SDS-PAGE analysis followed by Coomassie blue staining of pull-downs of mSTING-C-Terminal Domain (CTD) with 2’3’-cGAMP (+) or control (−) Sepharose. In all panels, data are representative of two independent experiments with similar results.
    Figure Legend Snippet: a , Sodium dodecyl sulfate (SDS)-PAGE analysis followed by Coomassie blue staining of His-tagged human SLC19A1 (Ni-NTA affinity-purified) pull-downs with Sepharose beads coupled with 2’3’-cGAMP (+) or control Sepharose beads (−). Input is shown in the right panel. b , Western blots of the samples in a with anti-SLC19A1 antibody. c , Pull-downs of SLC19A1 competed with CDNs. His-tagged SLC19A1 was incubated with no CDN or with the indicated competing CDNs (250 μM) before pulldowns with 2’3’-cGAMP-Sepharose, followed by SDS-PAGE and Western blotting with an anti-SLC19A1 antibody. A pulldown with control Sepharose is shown for comparison. For gel source data, see . d , SDS-PAGE analysis followed by Coomassie blue staining of pull-downs of mSTING-C-Terminal Domain (CTD) with 2’3’-cGAMP (+) or control (−) Sepharose. In all panels, data are representative of two independent experiments with similar results.

    Techniques Used: SDS Page, Staining, Affinity Purification, Control, Western Blot, Incubation, Comparison



    Similar Products

    rabbit ant human slc19a1 pab  (Boster Bio)
    90
    Boster Bio rabbit ant human slc19a1 pab
    <t>SLC19A1</t> is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 μg/ml) or 2’3’-cGAMP (15 μg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (10 μg/ml), or 3’3’-CDA (20 μg/ml). After 18-22h, tdTomato expression was quantified as in (a). c, Induction of IFNB mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 μg/ml 2’3’-RR CDA. d, Control THP-1 cells and SLC19A1–1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (15 μg/ml), or hIFN-β (100 ng/ml) and analyzed as in (a). e, Control THP-1 cells (n=7 clonal lines) and SLC19A1 −/− cells ( 19A1 −/− ; n=9 clonal lines) were exposed to 2’3’-RR CDA (2.22 μg/ml), 2’3’-cGAMP (10 μg/ml), and tdTomato reporter expression was analyzed as in (a). Mean ± SEM are shown. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated and analyzed as in (b). g, THP-1 cells were incubated with increasing concentrations of the competitive inhibitors methotrexate, 5-methyl tetrahydrofolate (5-me-THF) or DMSO as vehicle control, before stimulating with 2’3’-RR CDA (1.25 μg/ml), 2’3’-cGAMP (15 μg/ml) or hIFN-β (100 ng/ml). Cells were analyzed as in (a). For each stimulant, the data were normalized to the DMSO controls. In panels b-d and f-g, mean ± SEM of n=3 biological replicates are shown. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test for the comparison to stimulated control cells (b-d), unpaired two-tailed Student’s t tests for (e), or two-way ANOVA followed by uncorrected Fisher’s LSD tests (f). *a P = 0.0002; *b P = 0.0013; *c P = 0.0005; *d P =0.0006; **** P ≤ 0.0001; n.s. not significant.
    Rabbit Ant Human Slc19a1 Pab, supplied by Boster Bio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+ant+human+slc19a1+pab/pmc06785039-22-26-29?v=Boster+Bio
    Average 90 stars, based on 1 article reviews
    rabbit ant human slc19a1 pab - by Bioz Stars, 2026-08
    90/100 stars
    		  Buy from Supplier

    Image Search Results


    SLC19A1 is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 μg/ml) or 2’3’-cGAMP (15 μg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (10 μg/ml), or 3’3’-CDA (20 μg/ml). After 18-22h, tdTomato expression was quantified as in (a). c, Induction of IFNB mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 μg/ml 2’3’-RR CDA. d, Control THP-1 cells and SLC19A1–1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (15 μg/ml), or hIFN-β (100 ng/ml) and analyzed as in (a). e, Control THP-1 cells (n=7 clonal lines) and SLC19A1 −/− cells ( 19A1 −/− ; n=9 clonal lines) were exposed to 2’3’-RR CDA (2.22 μg/ml), 2’3’-cGAMP (10 μg/ml), and tdTomato reporter expression was analyzed as in (a). Mean ± SEM are shown. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated and analyzed as in (b). g, THP-1 cells were incubated with increasing concentrations of the competitive inhibitors methotrexate, 5-methyl tetrahydrofolate (5-me-THF) or DMSO as vehicle control, before stimulating with 2’3’-RR CDA (1.25 μg/ml), 2’3’-cGAMP (15 μg/ml) or hIFN-β (100 ng/ml). Cells were analyzed as in (a). For each stimulant, the data were normalized to the DMSO controls. In panels b-d and f-g, mean ± SEM of n=3 biological replicates are shown. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test for the comparison to stimulated control cells (b-d), unpaired two-tailed Student’s t tests for (e), or two-way ANOVA followed by uncorrected Fisher’s LSD tests (f). *a P = 0.0002; *b P = 0.0013; *c P = 0.0005; *d P =0.0006; **** P ≤ 0.0001; n.s. not significant.

    Journal: Nature

    Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

    doi: 10.1038/s41586-019-1553-0

    Figure Lengend Snippet: SLC19A1 is required for CDN-induced reporter expression. a, dCas9-KRAB-expressing THP-1 cells transduced with non-targeting gRNA (control), IRF3-1 gRNA or SLC19A1-1 gRNA were exposed to 2’3’-RR CDA (1.67 μg/ml) or 2’3’-cGAMP (15 μg/ml). 20h later, tdTomato expression was analyzed by flow cytometry. Representative dot plots of three independent experiments are shown. b, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (10 μg/ml), or 3’3’-CDA (20 μg/ml). After 18-22h, tdTomato expression was quantified as in (a). c, Induction of IFNB mRNA in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5h stimulation with 5 μg/ml 2’3’-RR CDA. d, Control THP-1 cells and SLC19A1–1 gRNA expressing THP-1 cells transduced with SLC19A1 (SLC. tr.) were stimulated with 2’3’-RR CDA (1.67 μg/ml), 2’3’-cGAMP (15 μg/ml), or hIFN-β (100 ng/ml) and analyzed as in (a). e, Control THP-1 cells (n=7 clonal lines) and SLC19A1 −/− cells ( 19A1 −/− ; n=9 clonal lines) were exposed to 2’3’-RR CDA (2.22 μg/ml), 2’3’-cGAMP (10 μg/ml), and tdTomato reporter expression was analyzed as in (a). Mean ± SEM are shown. f, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated and analyzed as in (b). g, THP-1 cells were incubated with increasing concentrations of the competitive inhibitors methotrexate, 5-methyl tetrahydrofolate (5-me-THF) or DMSO as vehicle control, before stimulating with 2’3’-RR CDA (1.25 μg/ml), 2’3’-cGAMP (15 μg/ml) or hIFN-β (100 ng/ml). Cells were analyzed as in (a). For each stimulant, the data were normalized to the DMSO controls. In panels b-d and f-g, mean ± SEM of n=3 biological replicates are shown. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post-test for the comparison to stimulated control cells (b-d), unpaired two-tailed Student’s t tests for (e), or two-way ANOVA followed by uncorrected Fisher’s LSD tests (f). *a P = 0.0002; *b P = 0.0013; *c P = 0.0005; *d P =0.0006; **** P ≤ 0.0001; n.s. not significant.

    Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

    Techniques: Expressing, Transduction, Control, Flow Cytometry, Plasmid Preparation, Incubation, Comparison, Two Tailed Test

    a , Thin layer chromatography (TLC) analysis of [ 32 P] ATP standard (STD) and enzymatically synthesized [ 32 P] 2’3’-cGAMP. 2’3’-cGAMP was purified on STING resin. Unbound nucleotides flowed through the resin (STING FT). Following four washes, the bound [32P] 2’3’-cGAMP was eluted over three fractions. b , DRaCALA binding analysis of [ 32 P] 2’3’-cGAMP to STING C-terminal domain (CTD) in the presence of competing unlabeled nucleotides (200 μM). c , Thin layer chromatography (TLC) analysis of [ 32 P]-ATP and enzymatically synthesized [ 32 P]-2’3’-cGAMP and [ 32 P]-c-di-AMP. d , Binding titration of [ 32 P] 2’3’-cGAMP or [ 32 P] c-di-AMP to mSTING C-Terminal Domain (CTD), determined with DRaCALA assays. Red dashed lines represent the 95% confidence interval for the non-linear regression. e , Time course of [ 32 P] 2’3’-cGAMP (left panel) or [ 32 P] 3’3’-CDA (right panel) uptake in THP-1 monocytes. f , TLC analysis (left panel) and STING-binding (DRaCALA) (right panel) of [ 32 P] ATP standard, or [ 32 P] 2’3’-cGAMP recovered from supernatants of THP-1 monocytes at the indicated time points. g , h , Effect of cell culture medium pH on [ 32 P] 2’3’-cGAMP uptake in THP-1 (g) monocytes or U937 monocytes (h). i , Time course of [ 32 P] 2’3’-cGAMP uptake by CIR cells transduced (tr.) with empty vector or SLC19A1 expression vector. j, mRNA expression levels of SLC19A1 (SLC.) in K562 cells expressing control shRNAs (sh1 and sh2) or an SLC19A1 -targeting shRNA (sh9). k, mRNA expression levels of CXCL10 in K562 cells described in panel j, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. l, [ 3 H]-Methotrexate uptake in K562 cells described in panel j, 1h after exposure to [ 3 H]-Methotrexate. m , Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells described in panel j. n , Time course of [ 32 P] 2’3’-cGAMP uptake in U937 monocytes in the presence or absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. o . Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells in the presence or absence of 500 μM competing, unlabeled (anti-) folates or sulfasalazine. p , Competition uptake assay of [ 32 P] 2’3’-cGAMP uptake in THP-1 cells in the presence of varying concentrations of competing, unlabeled 5-me-THF (IC50 = 4.10 ± 0.16 nM), methotrexate (IC50 = 54.83 ± 5.08 nM), 2’3’-cGAMP (IC50 = 1.89 ± 0.11 μM), sulfasalazine ((IC50 = 2.06 ± 0.17 μM), and folic acid (IC50 = 4.79 ± 0.08 μM). q , Trans-stimulation of [ 32 P] 2’3’-cGAMP influx in THP-1 cells by 5-me-THF. Cells were preloaded with indicated concentrations of 5-me-THF for 30 min. Cells were washed and incubated with [ 32 P] 2’3’-cGAMP for one hour. r , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated human PBMCs from four healthy donors. In panel a and c, data are representative of three independent experiments with similar results. In panel b, data are means of n=2 technical replicates and are representative of three independent experiments. In panel d and f, data are means of n=2 technical replicates and are representative of two independent experiments. In panel e and m, data are means ± SD of n=3 technical replicates and are representative of three independent experiments. In panel g, h, i, and n-r, data are means ± n=3 technical replicates and are representative of two independent experiments. In panel j and k, data are means ± SEM of n=3 biologically independent experiments. In panel l, data are means of n=2 biologically independent experiments.

    Journal: Nature

    Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

    doi: 10.1038/s41586-019-1553-0

    Figure Lengend Snippet: a , Thin layer chromatography (TLC) analysis of [ 32 P] ATP standard (STD) and enzymatically synthesized [ 32 P] 2’3’-cGAMP. 2’3’-cGAMP was purified on STING resin. Unbound nucleotides flowed through the resin (STING FT). Following four washes, the bound [32P] 2’3’-cGAMP was eluted over three fractions. b , DRaCALA binding analysis of [ 32 P] 2’3’-cGAMP to STING C-terminal domain (CTD) in the presence of competing unlabeled nucleotides (200 μM). c , Thin layer chromatography (TLC) analysis of [ 32 P]-ATP and enzymatically synthesized [ 32 P]-2’3’-cGAMP and [ 32 P]-c-di-AMP. d , Binding titration of [ 32 P] 2’3’-cGAMP or [ 32 P] c-di-AMP to mSTING C-Terminal Domain (CTD), determined with DRaCALA assays. Red dashed lines represent the 95% confidence interval for the non-linear regression. e , Time course of [ 32 P] 2’3’-cGAMP (left panel) or [ 32 P] 3’3’-CDA (right panel) uptake in THP-1 monocytes. f , TLC analysis (left panel) and STING-binding (DRaCALA) (right panel) of [ 32 P] ATP standard, or [ 32 P] 2’3’-cGAMP recovered from supernatants of THP-1 monocytes at the indicated time points. g , h , Effect of cell culture medium pH on [ 32 P] 2’3’-cGAMP uptake in THP-1 (g) monocytes or U937 monocytes (h). i , Time course of [ 32 P] 2’3’-cGAMP uptake by CIR cells transduced (tr.) with empty vector or SLC19A1 expression vector. j, mRNA expression levels of SLC19A1 (SLC.) in K562 cells expressing control shRNAs (sh1 and sh2) or an SLC19A1 -targeting shRNA (sh9). k, mRNA expression levels of CXCL10 in K562 cells described in panel j, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. l, [ 3 H]-Methotrexate uptake in K562 cells described in panel j, 1h after exposure to [ 3 H]-Methotrexate. m , Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells described in panel j. n , Time course of [ 32 P] 2’3’-cGAMP uptake in U937 monocytes in the presence or absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. o . Time course of [ 32 P] 2’3’-cGAMP uptake in K562 cells in the presence or absence of 500 μM competing, unlabeled (anti-) folates or sulfasalazine. p , Competition uptake assay of [ 32 P] 2’3’-cGAMP uptake in THP-1 cells in the presence of varying concentrations of competing, unlabeled 5-me-THF (IC50 = 4.10 ± 0.16 nM), methotrexate (IC50 = 54.83 ± 5.08 nM), 2’3’-cGAMP (IC50 = 1.89 ± 0.11 μM), sulfasalazine ((IC50 = 2.06 ± 0.17 μM), and folic acid (IC50 = 4.79 ± 0.08 μM). q , Trans-stimulation of [ 32 P] 2’3’-cGAMP influx in THP-1 cells by 5-me-THF. Cells were preloaded with indicated concentrations of 5-me-THF for 30 min. Cells were washed and incubated with [ 32 P] 2’3’-cGAMP for one hour. r , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated human PBMCs from four healthy donors. In panel a and c, data are representative of three independent experiments with similar results. In panel b, data are means of n=2 technical replicates and are representative of three independent experiments. In panel d and f, data are means of n=2 technical replicates and are representative of two independent experiments. In panel e and m, data are means ± SD of n=3 technical replicates and are representative of three independent experiments. In panel g, h, i, and n-r, data are means ± n=3 technical replicates and are representative of two independent experiments. In panel j and k, data are means ± SEM of n=3 biologically independent experiments. In panel l, data are means of n=2 biologically independent experiments.

    Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

    Techniques: Thin Layer Chromatography, Synthesized, Purification, Binding Assay, Titration, Cell Culture, Plasmid Preparation, Expressing, Control, shRNA, Incubation

    a, mRNA expression levels of Slc19a1 in mouse L1210 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh4 and sh6). b, mRNA expression levels of Cxcl10 in L1210 cells described in panel a stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. c, [ 3 H]-Methotrexate uptake in L1210 cells described in panel a 1h after exposure to [ 3 H]-Methotrexate. d, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells described in panel a. e, mRNA expression levels of Slc19a1 in mouse C1498 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh6). f, mRNA expression levels of Cxcl10 in the C1498 cells described in panel e, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. g, [ 3 H]-Methotrexate uptake in C1498 cells described in panel e 1h after exposure to [ 3 H]-Methotrexate. h , Time course of [ 32 P] 2’3’-cGAMP uptake in C1498 cells transduced with a non-targeting control shRNA (control) or Slc19a1 shRNA. i, j, mRNA expression levels of Slc19a1 in mouse bone marrow-derived macrophages (BMM) (i) or mouse bone marrow-derived dendritic cells (BMDCs) (j) transduced or not with control shRNAs (sh1 and 2) or an shRNA targeting Slc19a1 . k, l, mRNA expression of the Cxcl10 in cells described in panels i and j stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. m, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. n, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes pretreated or not with NHS-methotrexate (MTX) (5 μM). o, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells pretreated or not with NHS-MTX (5 μM). In panels a-c, e-g, j and l, data are means of n=2 biologically independent experiments. In panel d, h, i, k, n and o, data are means ± SD of n=3 technical replicates and are representative of two independent experiments. In panel m, data are means of n=2 technical replicates and are representative of two independent experiments. In time course experiments (d, h, m, n, o), data are presented as counts per minute (cpm) normalized to cell count.

    Journal: Nature

    Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

    doi: 10.1038/s41586-019-1553-0

    Figure Lengend Snippet: a, mRNA expression levels of Slc19a1 in mouse L1210 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh4 and sh6). b, mRNA expression levels of Cxcl10 in L1210 cells described in panel a stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. c, [ 3 H]-Methotrexate uptake in L1210 cells described in panel a 1h after exposure to [ 3 H]-Methotrexate. d, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells described in panel a. e, mRNA expression levels of Slc19a1 in mouse C1498 cells expressing control shRNAs (sh1 and sh2) or Slc19a1 -targeting shRNA (sh6). f, mRNA expression levels of Cxcl10 in the C1498 cells described in panel e, stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. g, [ 3 H]-Methotrexate uptake in C1498 cells described in panel e 1h after exposure to [ 3 H]-Methotrexate. h , Time course of [ 32 P] 2’3’-cGAMP uptake in C1498 cells transduced with a non-targeting control shRNA (control) or Slc19a1 shRNA. i, j, mRNA expression levels of Slc19a1 in mouse bone marrow-derived macrophages (BMM) (i) or mouse bone marrow-derived dendritic cells (BMDCs) (j) transduced or not with control shRNAs (sh1 and 2) or an shRNA targeting Slc19a1 . k, l, mRNA expression of the Cxcl10 in cells described in panels i and j stimulated with 5 μg/ml 2’3’-RR CDA (CDN) for 5h. m, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. n, Time course of [ 32 P] 2’3’-cGAMP uptake in primary murine splenocytes pretreated or not with NHS-methotrexate (MTX) (5 μM). o, Time course of [ 32 P] 2’3’-cGAMP uptake in L1210 cells pretreated or not with NHS-MTX (5 μM). In panels a-c, e-g, j and l, data are means of n=2 biologically independent experiments. In panel d, h, i, k, n and o, data are means ± SD of n=3 technical replicates and are representative of two independent experiments. In panel m, data are means of n=2 technical replicates and are representative of two independent experiments. In time course experiments (d, h, m, n, o), data are presented as counts per minute (cpm) normalized to cell count.

    Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

    Techniques: Expressing, Control, shRNA, Transduction, Derivative Assay, Cell Counting

    SLC19A1 transports CDNs. a , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control CRISPRi gRNA or SLC19A1 CRISPRi gRNA (right panel). b , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by K562 cells transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control shRNA (control) or SLC19A1 shRNA (right panel). c , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated THP-1 (left panel) and K562 (right panel) cells. d , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes in the presence and absence of 100 μM competing, unlabeled cyclic dinucleotides (left panel) or 200 μM competing, unlabeled nucleotides (right panel). e , Time course of [ 32 P] 2’3’-cGAMP uptake in THP-1 monocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. f , Normalized [ 32 P] 2’3’-cGAMP uptake after three hours in human PBMCs from six healthy donors in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. g , Coomassie staining and western blot analysis of pulldowns by 2’3’-cGAMP (+) or control (−) Sepharose of mSTING-CTD or hSLC19A1. h , Western blot analysis of hSLC19A1 affinity purification (AP) with 2’3’-cGAMP Sepharose in the absence (−) or presence (+) of free, unbound 2’3’-cGAMP, 5-me-THF, or methotrexate (250 μM). In panels a-d, data are means ± SEM of n=3 biological replicates. In panel e, data are means ± SD of n=3 technical replicates and data are representative of three independent experiments. In panel f, data are means ± SD of n=6 healthy donors conducted over two independent experiments. Panels g and h are representative of two independent experiments; for gel source data, see . Statistical analyses were performed using unpaired, two-tailed Student’s t-tests (a-d), or a one-way ANOVA followed by a Tukey’s post-test (f). **** P ≤ 0.0001.

    Journal: Nature

    Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

    doi: 10.1038/s41586-019-1553-0

    Figure Lengend Snippet: SLC19A1 transports CDNs. a , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control CRISPRi gRNA or SLC19A1 CRISPRi gRNA (right panel). b , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by K562 cells transduced with empty vector (control) or SLC19A1 expression vector (left panel), or transduced with a non-targeting control shRNA (control) or SLC19A1 shRNA (right panel). c , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour in DMSO or NHS-methotrexate (MTX) (5 μM) treated THP-1 (left panel) and K562 (right panel) cells. d , Normalized [ 32 P] 2’3’-cGAMP uptake after one hour by THP-1 monocytes in the presence and absence of 100 μM competing, unlabeled cyclic dinucleotides (left panel) or 200 μM competing, unlabeled nucleotides (right panel). e , Time course of [ 32 P] 2’3’-cGAMP uptake in THP-1 monocytes in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. f , Normalized [ 32 P] 2’3’-cGAMP uptake after three hours in human PBMCs from six healthy donors in the presence and absence of 500 μM competing, unlabeled (anti-) folates and sulfasalazine. g , Coomassie staining and western blot analysis of pulldowns by 2’3’-cGAMP (+) or control (−) Sepharose of mSTING-CTD or hSLC19A1. h , Western blot analysis of hSLC19A1 affinity purification (AP) with 2’3’-cGAMP Sepharose in the absence (−) or presence (+) of free, unbound 2’3’-cGAMP, 5-me-THF, or methotrexate (250 μM). In panels a-d, data are means ± SEM of n=3 biological replicates. In panel e, data are means ± SD of n=3 technical replicates and data are representative of three independent experiments. In panel f, data are means ± SD of n=6 healthy donors conducted over two independent experiments. Panels g and h are representative of two independent experiments; for gel source data, see . Statistical analyses were performed using unpaired, two-tailed Student’s t-tests (a-d), or a one-way ANOVA followed by a Tukey’s post-test (f). **** P ≤ 0.0001.

    Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

    Techniques: Transduction, Plasmid Preparation, Control, Expressing, shRNA, Staining, Western Blot, Affinity Purification, Two Tailed Test

    a , Sodium dodecyl sulfate (SDS)-PAGE analysis followed by Coomassie blue staining of His-tagged human SLC19A1 (Ni-NTA affinity-purified) pull-downs with Sepharose beads coupled with 2’3’-cGAMP (+) or control Sepharose beads (−). Input is shown in the right panel. b , Western blots of the samples in a with anti-SLC19A1 antibody. c , Pull-downs of SLC19A1 competed with CDNs. His-tagged SLC19A1 was incubated with no CDN or with the indicated competing CDNs (250 μM) before pulldowns with 2’3’-cGAMP-Sepharose, followed by SDS-PAGE and Western blotting with an anti-SLC19A1 antibody. A pulldown with control Sepharose is shown for comparison. For gel source data, see . d , SDS-PAGE analysis followed by Coomassie blue staining of pull-downs of mSTING-C-Terminal Domain (CTD) with 2’3’-cGAMP (+) or control (−) Sepharose. In all panels, data are representative of two independent experiments with similar results.

    Journal: Nature

    Article Title: SLC19A1 transports immunoreactive cyclic dinucleotides

    doi: 10.1038/s41586-019-1553-0

    Figure Lengend Snippet: a , Sodium dodecyl sulfate (SDS)-PAGE analysis followed by Coomassie blue staining of His-tagged human SLC19A1 (Ni-NTA affinity-purified) pull-downs with Sepharose beads coupled with 2’3’-cGAMP (+) or control Sepharose beads (−). Input is shown in the right panel. b , Western blots of the samples in a with anti-SLC19A1 antibody. c , Pull-downs of SLC19A1 competed with CDNs. His-tagged SLC19A1 was incubated with no CDN or with the indicated competing CDNs (250 μM) before pulldowns with 2’3’-cGAMP-Sepharose, followed by SDS-PAGE and Western blotting with an anti-SLC19A1 antibody. A pulldown with control Sepharose is shown for comparison. For gel source data, see . d , SDS-PAGE analysis followed by Coomassie blue staining of pull-downs of mSTING-C-Terminal Domain (CTD) with 2’3’-cGAMP (+) or control (−) Sepharose. In all panels, data are representative of two independent experiments with similar results.

    Article Snippet: Other antibodies: rabbit-anti-human IRF3 mAb (Abcam, cat. #: EP2419Y, used 1:2000 for IB), mouse-anti-human transferrin receptor mAb (Thermo Fischer Scientific, clone H68.4, used 1:1000 for IB), rabbit-ant-human SLC19A1 pAb (BosterBio, cat. #: PB9504, used 0.4 μg/ml for IB), APC-conjugated mouse-anti-human CD55 mAb (BioLegend, clone JS11, used 1:50 for flow cytometry), mouse-anti-human CD59 mAb (BioLegend clone p282, used 1:250 for flow cytometry), APC-conjugated goat-anti-mouse IgG (BioLegend, cat. #: 405308, used 1:100 for flow cytometry).

    Techniques: SDS Page, Staining, Affinity Purification, Control, Western Blot, Incubation, Comparison