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  • 99
    Thermo Fisher ctp
    Ctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 180 article reviews
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    ctp - by Bioz Stars, 2020-04
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    99
    Millipore ctp
    Sensitivity of full-length RNA products to alkaline phosphatase treatment Full-length RNA products of in vitro transcription reactions with [γ 32 <t>P]-ATP</t> or [α 32 P]-NAD + as initiating nucleotide and <t>CTP,</t> GTP, and UTP as extending nucleotides ( E. coli RNAP; P rnaI fused to an A-less cassette ). Products were treated with alkaline phosphatase (AP; processes 5′ phosphates) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. Results indicate that full-length RNA products generated in reactions with [α 32 P]-NAD + as initiating nucleotide are not sensitive to AP until they are processed by NudC. M, 100-nt marker. For gel source data, see Supplementary Figure 1 .
    Ctp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cytidine triphosphate ctp
    Sensitivity of full-length RNA products to alkaline phosphatase treatment Full-length RNA products of in vitro transcription reactions with [γ 32 <t>P]-ATP</t> or [α 32 P]-NAD + as initiating nucleotide and <t>CTP,</t> GTP, and UTP as extending nucleotides ( E. coli RNAP; P rnaI fused to an A-less cassette ). Products were treated with alkaline phosphatase (AP; processes 5′ phosphates) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. Results indicate that full-length RNA products generated in reactions with [α 32 P]-NAD + as initiating nucleotide are not sensitive to AP until they are processed by NudC. M, 100-nt marker. For gel source data, see Supplementary Figure 1 .
    Cytidine Triphosphate Ctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche cytidine triphosphate ctp
    Template-directed oligoribonucleotide synthesis catalyzed by T7 DNA primase. ( a ) The reaction contained the oligonucleotide 5′-GGGTCA 10 -3′ with the primase recognition sequence and [α- 33 <t>P]-CTP</t> and <t>ATP</t> in the standard reaction mixture in the presence of 8 mM MgCl 2 or 8 mM MnCl 2 in a reaction buffer containing increasing amounts of T7 DNA primase (0, 1.11, 3.33, and 10 µM). After incubation for 20 min at RT, the radioactive products were analyzed by electrophoresis through a 25% polyacrylamide gel containing 7 M urea and visualized using autoradiography. ( b ) Quantification of primase activity. The bands in the gel presented in a were analyzed using autoradiography.
    Cytidine Triphosphate Ctp, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    GE Healthcare cytidine triphosphate ctp
    HPLC profiles of T7 DNA primase reaction mixtures using DNA template CCCC GGGT CT 25 . Crude enzymatic mixtures were analyzed directly. Reaction conditions were as follows: T7 DNA primase reaction buffer, <t>DTT</t> and BSA as given in Material and Methods, 5 mM <t>CTP,</t> 1 mM GpppA (A) or 7Me GpppA (B) , 10 µM DNA template CCCC GGGT CT 25 (named dAC3) and 4 µM T7 DNA primase. Reactions were incubated at 37°C for 6 h. The first section (marked in gray) denotes the removal of proteic material and remaining CTP by an on-line cleaning procedure using a pre-column. The produced capped RNAs were then separated on the reverse-phase C18 column. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min, reaching 30% after 35 min and 50% after 40 min.
    Cytidine Triphosphate Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 81/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega cytidine triphosphate ctp
    HPLC profiles of T7 DNA primase reaction mixtures using DNA template CCCC GGGT CT 25 . Crude enzymatic mixtures were analyzed directly. Reaction conditions were as follows: T7 DNA primase reaction buffer, <t>DTT</t> and BSA as given in Material and Methods, 5 mM <t>CTP,</t> 1 mM GpppA (A) or 7Me GpppA (B) , 10 µM DNA template CCCC GGGT CT 25 (named dAC3) and 4 µM T7 DNA primase. Reactions were incubated at 37°C for 6 h. The first section (marked in gray) denotes the removal of proteic material and remaining CTP by an on-line cleaning procedure using a pre-column. The produced capped RNAs were then separated on the reverse-phase C18 column. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min, reaching 30% after 35 min and 50% after 40 min.
    Cytidine Triphosphate Ctp, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene cytidine triphosphate ctp 32
    HPLC profiles of T7 DNA primase reaction mixtures using DNA template CCCC GGGT CT 25 . Crude enzymatic mixtures were analyzed directly. Reaction conditions were as follows: T7 DNA primase reaction buffer, <t>DTT</t> and BSA as given in Material and Methods, 5 mM <t>CTP,</t> 1 mM GpppA (A) or 7Me GpppA (B) , 10 µM DNA template CCCC GGGT CT 25 (named dAC3) and 4 µM T7 DNA primase. Reactions were incubated at 37°C for 6 h. The first section (marked in gray) denotes the removal of proteic material and remaining CTP by an on-line cleaning procedure using a pre-column. The produced capped RNAs were then separated on the reverse-phase C18 column. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min, reaching 30% after 35 min and 50% after 40 min.
    Cytidine Triphosphate Ctp 32, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer cy3 cytidine triphosphate
    Transfection of Cells with Liposomes for Induced Expression of Different Proteins (A) The schematic overview shows the process of mRNA incorporation in liposomes and subsequent transfection and mRNA translation. During transfection, the lipoplexes are endocytosed by the cells of interest. Inside the cell, the lipid layer is degraded and mRNA is released. In the cytosol, the mRNA is translated by ribosomes and the protein is subsequently released intra- or extracellularly. (B) The presence of <t>Cy3-labeled</t> EGFP-encoding mRNA after liposome uptake and expression of EGFP protein in cells 24 hr posttransfection.
    Cy3 Cytidine Triphosphate, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    GE Healthcare α32 p ctp
    Transfection of Cells with Liposomes for Induced Expression of Different Proteins (A) The schematic overview shows the process of mRNA incorporation in liposomes and subsequent transfection and mRNA translation. During transfection, the lipoplexes are endocytosed by the cells of interest. Inside the cell, the lipid layer is degraded and mRNA is released. In the cytosol, the mRNA is translated by ribosomes and the protein is subsequently released intra- or extracellularly. (B) The presence of <t>Cy3-labeled</t> EGFP-encoding mRNA after liposome uptake and expression of EGFP protein in cells 24 hr posttransfection.
    α32 P Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 84/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    NEN Life Science cytidine triphosphate
    Transfection of Cells with Liposomes for Induced Expression of Different Proteins (A) The schematic overview shows the process of mRNA incorporation in liposomes and subsequent transfection and mRNA translation. During transfection, the lipoplexes are endocytosed by the cells of interest. Inside the cell, the lipid layer is degraded and mRNA is released. In the cytosol, the mRNA is translated by ribosomes and the protein is subsequently released intra- or extracellularly. (B) The presence of <t>Cy3-labeled</t> EGFP-encoding mRNA after liposome uptake and expression of EGFP protein in cells 24 hr posttransfection.
    Cytidine Triphosphate, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 81/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Valiant α 32 p cytidine triphosphate ctp
    Transfection of Cells with Liposomes for Induced Expression of Different Proteins (A) The schematic overview shows the process of mRNA incorporation in liposomes and subsequent transfection and mRNA translation. During transfection, the lipoplexes are endocytosed by the cells of interest. Inside the cell, the lipid layer is degraded and mRNA is released. In the cytosol, the mRNA is translated by ribosomes and the protein is subsequently released intra- or extracellularly. (B) The presence of <t>Cy3-labeled</t> EGFP-encoding mRNA after liposome uptake and expression of EGFP protein in cells 24 hr posttransfection.
    α 32 P Cytidine Triphosphate Ctp, supplied by Valiant, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Agilent technologies cyanine 3 cytidine triphosphate
    Transfection of Cells with Liposomes for Induced Expression of Different Proteins (A) The schematic overview shows the process of mRNA incorporation in liposomes and subsequent transfection and mRNA translation. During transfection, the lipoplexes are endocytosed by the cells of interest. Inside the cell, the lipid layer is degraded and mRNA is released. In the cytosol, the mRNA is translated by ribosomes and the protein is subsequently released intra- or extracellularly. (B) The presence of <t>Cy3-labeled</t> EGFP-encoding mRNA after liposome uptake and expression of EGFP protein in cells 24 hr posttransfection.
    Cyanine 3 Cytidine Triphosphate, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 98/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PerkinElmer α 32 p ctp
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    α 32 P Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ctp inhibitor
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    Ctp Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Jena Bioscience tnp ctp
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    Tnp Ctp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Jena Bioscience ctp
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    Ctp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 98/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ctp/product/Jena Bioscience
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    99
    PerkinElmer cyanine 5 ctp
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    Cyanine 5 Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 181 article reviews
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    99
    PerkinElmer ctp α 32p
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    Ctp α 32p, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher cy3 ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    Cy3 Ctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Concert Pharmaceuticals ctp 656
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    Ctp 656, supplied by Concert Pharmaceuticals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    DuPont de Nemours p ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    P Ctp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 84/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    GE Healthcare p ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    P Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 87/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NEN Life Science p ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
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    PerkinElmer p32 ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
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    PerkinElmer s35 ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    S35 Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare 35s ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    35s Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Enzo Biochem bio ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    Bio Ctp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies cy3 ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    Cy3 Ctp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 590 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare cy3 ctp
    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 <t>P-CTP</t> for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.
    Cy3 Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Sensitivity of full-length RNA products to alkaline phosphatase treatment Full-length RNA products of in vitro transcription reactions with [γ 32 P]-ATP or [α 32 P]-NAD + as initiating nucleotide and CTP, GTP, and UTP as extending nucleotides ( E. coli RNAP; P rnaI fused to an A-less cassette ). Products were treated with alkaline phosphatase (AP; processes 5′ phosphates) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. Results indicate that full-length RNA products generated in reactions with [α 32 P]-NAD + as initiating nucleotide are not sensitive to AP until they are processed by NudC. M, 100-nt marker. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA

    doi: 10.1038/nature18622

    Figure Lengend Snippet: Sensitivity of full-length RNA products to alkaline phosphatase treatment Full-length RNA products of in vitro transcription reactions with [γ 32 P]-ATP or [α 32 P]-NAD + as initiating nucleotide and CTP, GTP, and UTP as extending nucleotides ( E. coli RNAP; P rnaI fused to an A-less cassette ). Products were treated with alkaline phosphatase (AP; processes 5′ phosphates) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. Results indicate that full-length RNA products generated in reactions with [α 32 P]-NAD + as initiating nucleotide are not sensitive to AP until they are processed by NudC. M, 100-nt marker. For gel source data, see Supplementary Figure 1 .

    Article Snippet: ATP and CTP (Sigma-Aldrich) were soaked into RPo crystals by addition of 0.2 μl 40 mM ATP and 40 mM CTP in 60% (v/v) RB to the crystallization drop, and incubated for 2 min at 22 °C.

    Techniques: In Vitro, Generated, Marker

    NCIN-mediated transcription initiation in vivo a. Templates having rnaI , T7A1 , and rnaI (-1C) promoters fused to identical transcribed regions (promoter elements, start sites, and position of RNA 3′-end in gray; DNA that directs synthesis of reference RNA in blue; site for MazF-mt3 endoribonuclease used to generate RNA products having uniform RNA 3′-ends, underlined). b. NCIN capping in vitro (left; 1 mM NAD + , 200 μM ATP, CTP, UTP, and GTP) and in vivo (right; RNA isolated from cells, treated with MazF-mt3 or MazF-mt3 plus NudC, and detected by hybridization). M, markers (40-nt, 50-nt). c. Effects of NCIN capping on RNA stability in vivo ( rnaI template; left, exponential-phase cells; right, stationary-phase cells; times, minutes after addition of RNA-synthesis inhibitor rifampin; half-life values are the mean±SEM of 3 determinations for exponential phase and 5 determinations for stationary phase). For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA

    doi: 10.1038/nature18622

    Figure Lengend Snippet: NCIN-mediated transcription initiation in vivo a. Templates having rnaI , T7A1 , and rnaI (-1C) promoters fused to identical transcribed regions (promoter elements, start sites, and position of RNA 3′-end in gray; DNA that directs synthesis of reference RNA in blue; site for MazF-mt3 endoribonuclease used to generate RNA products having uniform RNA 3′-ends, underlined). b. NCIN capping in vitro (left; 1 mM NAD + , 200 μM ATP, CTP, UTP, and GTP) and in vivo (right; RNA isolated from cells, treated with MazF-mt3 or MazF-mt3 plus NudC, and detected by hybridization). M, markers (40-nt, 50-nt). c. Effects of NCIN capping on RNA stability in vivo ( rnaI template; left, exponential-phase cells; right, stationary-phase cells; times, minutes after addition of RNA-synthesis inhibitor rifampin; half-life values are the mean±SEM of 3 determinations for exponential phase and 5 determinations for stationary phase). For gel source data, see Supplementary Figure 1 .

    Article Snippet: ATP and CTP (Sigma-Aldrich) were soaked into RPo crystals by addition of 0.2 μl 40 mM ATP and 40 mM CTP in 60% (v/v) RB to the crystallization drop, and incubated for 2 min at 22 °C.

    Techniques: In Vivo, In Vitro, Isolation, Hybridization

    De novo transcription initiation by ATP and NCINs a. Structures of ATP, NAD + , NADH, and dpCoA. Red, identical atoms. b. Initial RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP as extending nucleotide ( E. coli RNAP; P rnaI ; see analogous data for P gadY in Figure 1b ). Products were treated with RppH (processes 5′-triphosphate RNA to 5′-monophosphate RNA and 5′-NTP to 5′-NDP/5′-NMP 9 , 14 ) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA

    doi: 10.1038/nature18622

    Figure Lengend Snippet: De novo transcription initiation by ATP and NCINs a. Structures of ATP, NAD + , NADH, and dpCoA. Red, identical atoms. b. Initial RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP as extending nucleotide ( E. coli RNAP; P rnaI ; see analogous data for P gadY in Figure 1b ). Products were treated with RppH (processes 5′-triphosphate RNA to 5′-monophosphate RNA and 5′-NTP to 5′-NDP/5′-NMP 9 , 14 ) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. For gel source data, see Supplementary Figure 1 .

    Article Snippet: ATP and CTP (Sigma-Aldrich) were soaked into RPo crystals by addition of 0.2 μl 40 mM ATP and 40 mM CTP in 60% (v/v) RB to the crystallization drop, and incubated for 2 min at 22 °C.

    Techniques: In Vitro

    Promoter-sequence effects on efficiency of NCIN-mediated transcription initiation a. NCIN capping requires A +1 . Top, promoters of NAD + -capped RNAs (promoter elements and start sites in gray). Bottom, initial RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP as extending nucleotide [ E. coli RNAP; wt (+1A), P rnaI (upper) or P gadY (lower); mut (+1G), +1G derivative of P rnaI (upper) or P gadY (lower)]. b. Promoter sequence determinants in addition to A +1 affect NCIN capping. Top, control +1A promoters. Bottom left, dependence of NAD + capping on [NAD + ]/[ATP] ratio (mean±SEM of 4 determinations). Bottom right, relative efficiencies of NAD + capping. c. Promoter position −1 affects NCIN capping. Top, P rnaI (-1C) (-1 in black). Other features as in b . (mean±SEM of 3 determinations). For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA

    doi: 10.1038/nature18622

    Figure Lengend Snippet: Promoter-sequence effects on efficiency of NCIN-mediated transcription initiation a. NCIN capping requires A +1 . Top, promoters of NAD + -capped RNAs (promoter elements and start sites in gray). Bottom, initial RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP as extending nucleotide [ E. coli RNAP; wt (+1A), P rnaI (upper) or P gadY (lower); mut (+1G), +1G derivative of P rnaI (upper) or P gadY (lower)]. b. Promoter sequence determinants in addition to A +1 affect NCIN capping. Top, control +1A promoters. Bottom left, dependence of NAD + capping on [NAD + ]/[ATP] ratio (mean±SEM of 4 determinations). Bottom right, relative efficiencies of NAD + capping. c. Promoter position −1 affects NCIN capping. Top, P rnaI (-1C) (-1 in black). Other features as in b . (mean±SEM of 3 determinations). For gel source data, see Supplementary Figure 1 .

    Article Snippet: ATP and CTP (Sigma-Aldrich) were soaked into RPo crystals by addition of 0.2 μl 40 mM ATP and 40 mM CTP in 60% (v/v) RB to the crystallization drop, and incubated for 2 min at 22 °C.

    Techniques: Sequencing, In Vitro

    De novo transcription initiation by ATP and NCINs a. Structures of ATP, NAD + , NADH, and dpCoA. Red, identical atoms. b. Initial RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP as extending nucleotide ( E. coli RNAP; P gadY ). Products were treated with RppH (processes 5′-triphosphate RNA to 5′-monophosphate RNA and 5′-NTP to 5′-NDP/5′-NMP 9 , 14 ) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. c, d. Full-length RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP, GTP, and UTP as extending nucleotides (c), or with [γ 32 P]-ATP or [α 32 P]-NAD + as initiating nucleotide and CTP, GTP, and UTP as extending nucleotides (d) ( E. coli RNAP; P rnaI fused to an A-less cassette ). M, 100-nt marker. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA

    doi: 10.1038/nature18622

    Figure Lengend Snippet: De novo transcription initiation by ATP and NCINs a. Structures of ATP, NAD + , NADH, and dpCoA. Red, identical atoms. b. Initial RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP as extending nucleotide ( E. coli RNAP; P gadY ). Products were treated with RppH (processes 5′-triphosphate RNA to 5′-monophosphate RNA and 5′-NTP to 5′-NDP/5′-NMP 9 , 14 ) or NudC (processes 5′-NAD + /NADH-capped RNA to 5′-monophosphate RNA 6 ) as indicated. c, d. Full-length RNA products of in vitro transcription reactions with ATP, NAD + , NADH, or dpCoA as initiating nucleotide and [α 32 P]-CTP, GTP, and UTP as extending nucleotides (c), or with [γ 32 P]-ATP or [α 32 P]-NAD + as initiating nucleotide and CTP, GTP, and UTP as extending nucleotides (d) ( E. coli RNAP; P rnaI fused to an A-less cassette ). M, 100-nt marker. For gel source data, see Supplementary Figure 1 .

    Article Snippet: ATP and CTP (Sigma-Aldrich) were soaked into RPo crystals by addition of 0.2 μl 40 mM ATP and 40 mM CTP in 60% (v/v) RB to the crystallization drop, and incubated for 2 min at 22 °C.

    Techniques: In Vitro, Marker

    Promoter-sequence effects on efficiency of NCIN-mediated transcription initiation: NAD + a. Templates having rnaI , gadY , N25 , and T7A1 promoters used in the assays. b . Representative raw data from experiments of Figure 2b . Initial RNA products of in vitro transcription reactions performed in the presence of 50 μM ATP and 1 mM NAD + as initiating nucleotides and [α 32 P]-CTP as extending nucleotide ( E. coli RNAP; P rnaI , P gadY , P N25 , or P T7A1 ). (We note that contaminating AMP in the NAD + stock results in production of pAp*C.) For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA

    doi: 10.1038/nature18622

    Figure Lengend Snippet: Promoter-sequence effects on efficiency of NCIN-mediated transcription initiation: NAD + a. Templates having rnaI , gadY , N25 , and T7A1 promoters used in the assays. b . Representative raw data from experiments of Figure 2b . Initial RNA products of in vitro transcription reactions performed in the presence of 50 μM ATP and 1 mM NAD + as initiating nucleotides and [α 32 P]-CTP as extending nucleotide ( E. coli RNAP; P rnaI , P gadY , P N25 , or P T7A1 ). (We note that contaminating AMP in the NAD + stock results in production of pAp*C.) For gel source data, see Supplementary Figure 1 .

    Article Snippet: ATP and CTP (Sigma-Aldrich) were soaked into RPo crystals by addition of 0.2 μl 40 mM ATP and 40 mM CTP in 60% (v/v) RB to the crystallization drop, and incubated for 2 min at 22 °C.

    Techniques: Sequencing, In Vitro

    Inhibition of PfIspD by 1 R ,3 S -MMV008138 is competitive with CTP but not MEP. (a) Kinetic parameters with respect to the CTP substrate. Each data point represents mean ± SEM from at least three independent experiments. (b) Lineweaver–Burk double-reciprocal plots of wild-type PfIspD activity over a range of CTP substrate concentrations, for illustrative purposes only. (c) Kinetic parameters with respect to the MEP substrate. Each data point represents the mean ± SEM from at least three independent experiments. (d) Lineweaver–Burk double-reciprocal plots of wild-type PfIspD activity over a range of MEP substrate concentrations, for illustrative purposes only.

    Journal: ACS infectious diseases

    Article Title: Plasmodium IspD (2-C-Methyl-D-erythritol 4-Phosphate Cytidyltransferase), an Essential and Druggable Antimalarial Target

    doi: 10.1021/id500047s

    Figure Lengend Snippet: Inhibition of PfIspD by 1 R ,3 S -MMV008138 is competitive with CTP but not MEP. (a) Kinetic parameters with respect to the CTP substrate. Each data point represents mean ± SEM from at least three independent experiments. (b) Lineweaver–Burk double-reciprocal plots of wild-type PfIspD activity over a range of CTP substrate concentrations, for illustrative purposes only. (c) Kinetic parameters with respect to the MEP substrate. Each data point represents the mean ± SEM from at least three independent experiments. (d) Lineweaver–Burk double-reciprocal plots of wild-type PfIspD activity over a range of MEP substrate concentrations, for illustrative purposes only.

    Article Snippet: IC50 assays contained 50 μ M CTP (Sigma), 500 μ M MEP (Echelon Biosciences), 0.1% DMSO (vehicle for 1 R ,3 S -MMV008138), and 0.2 mM MESG.

    Techniques: Inhibition, Activity Assay

    β IV -spectrin is a CaMKII-binding protein in heart. ( A ) β IV -spectrin contains an N-terminal actin-binding domain (NTD), 17 spectrin repeats, and specific and C-terminal domains (SD/CTD). The putative CaMKII-binding site is denoted by an asterisk. ( B ) The putative CaMKII-binding domain in β IV -spectrin was homologous to a CaMKIIδ autoregulatory domain motif and conserved across orthologs. ( C ) CTP-P bound radiolabeled CaMKIIδ; CTP-C and GST beads alone lacked binding. ( D ) β IV -spectrin RNA levels in adult rat brain and heart. ( E ) β IV -spectrin (Σ1 and Σ6) in ventricular lysates from multiple species. β IV -spectrin was expressed approximately 8–10 fold higher in cerebellum than in heart. Cardiac β IV -spectrin migrated approximately 4 kDa larger than did cerebellar β IV -spectrin. ( F ) CTP-P, but not CTP-C, associated with CaMKIIδ from rat heart. ( G ) Endogenous CaMKIIδ and β IV -spectrin coimmunoprecipitated from adult heart lysate. ( H and I ) β IV -spectrin and N-cadherin in rat cardiomyocytes. Nuclei are shown by Topro-3 dye (blue). ( J ) CaMKIIδ localization in adult rat myocytes. CaMKIIδ localized to the intercalated disc (white arrows) here and to a second population at transverse-tubules (yellow arrows). Ventricular sections stained for ( K ) β IV -spectrin, ( L ) N-cadherin, and ( M ) CaMKIIδ showed coexpression of these proteins at the intercalated disc (white arrows). ( N and P ) Ankyrin-G and ( O and Q ) Na v 1.5 were also found at the intercalated disc (white arrows) in rat myocytes and tissue sections. ( R – U ) Coimmunoprecipitation studies demonstrate cardiac complex of β IV -spectrin, CaMKIIδ, ankyrin-G (AnkG), and Na v 1.5. Scale bars: 10 μm ( H – Q ).

    Journal: The Journal of Clinical Investigation

    Article Title: A ?IV-spectrin/CaMKII signaling complex is essential for membrane excitability in mice

    doi: 10.1172/JCI43621

    Figure Lengend Snippet: β IV -spectrin is a CaMKII-binding protein in heart. ( A ) β IV -spectrin contains an N-terminal actin-binding domain (NTD), 17 spectrin repeats, and specific and C-terminal domains (SD/CTD). The putative CaMKII-binding site is denoted by an asterisk. ( B ) The putative CaMKII-binding domain in β IV -spectrin was homologous to a CaMKIIδ autoregulatory domain motif and conserved across orthologs. ( C ) CTP-P bound radiolabeled CaMKIIδ; CTP-C and GST beads alone lacked binding. ( D ) β IV -spectrin RNA levels in adult rat brain and heart. ( E ) β IV -spectrin (Σ1 and Σ6) in ventricular lysates from multiple species. β IV -spectrin was expressed approximately 8–10 fold higher in cerebellum than in heart. Cardiac β IV -spectrin migrated approximately 4 kDa larger than did cerebellar β IV -spectrin. ( F ) CTP-P, but not CTP-C, associated with CaMKIIδ from rat heart. ( G ) Endogenous CaMKIIδ and β IV -spectrin coimmunoprecipitated from adult heart lysate. ( H and I ) β IV -spectrin and N-cadherin in rat cardiomyocytes. Nuclei are shown by Topro-3 dye (blue). ( J ) CaMKIIδ localization in adult rat myocytes. CaMKIIδ localized to the intercalated disc (white arrows) here and to a second population at transverse-tubules (yellow arrows). Ventricular sections stained for ( K ) β IV -spectrin, ( L ) N-cadherin, and ( M ) CaMKIIδ showed coexpression of these proteins at the intercalated disc (white arrows). ( N and P ) Ankyrin-G and ( O and Q ) Na v 1.5 were also found at the intercalated disc (white arrows) in rat myocytes and tissue sections. ( R – U ) Coimmunoprecipitation studies demonstrate cardiac complex of β IV -spectrin, CaMKIIδ, ankyrin-G (AnkG), and Na v 1.5. Scale bars: 10 μm ( H – Q ).

    Article Snippet: [35 S]-labeled CaMKIIδ was incubated with CTP-P, a biotinylated peptide mimicking the putative kinase-binding domain in human βIV -spectrin, or CTP-C, a scrambled peptide, in 500 μl binding buffer (50 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA; 150 mM NaCl; 0.1% Triton X-100; and 1:1,000 protease inhibitor [Sigma-Aldrich]) overnight at 4°C.

    Techniques: Binding Assay, Staining

    Template-directed oligoribonucleotide synthesis catalyzed by T7 DNA primase. ( a ) The reaction contained the oligonucleotide 5′-GGGTCA 10 -3′ with the primase recognition sequence and [α- 33 P]-CTP and ATP in the standard reaction mixture in the presence of 8 mM MgCl 2 or 8 mM MnCl 2 in a reaction buffer containing increasing amounts of T7 DNA primase (0, 1.11, 3.33, and 10 µM). After incubation for 20 min at RT, the radioactive products were analyzed by electrophoresis through a 25% polyacrylamide gel containing 7 M urea and visualized using autoradiography. ( b ) Quantification of primase activity. The bands in the gel presented in a were analyzed using autoradiography.

    Journal: Scientific Reports

    Article Title: Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase

    doi: 10.1038/s41598-017-05534-3

    Figure Lengend Snippet: Template-directed oligoribonucleotide synthesis catalyzed by T7 DNA primase. ( a ) The reaction contained the oligonucleotide 5′-GGGTCA 10 -3′ with the primase recognition sequence and [α- 33 P]-CTP and ATP in the standard reaction mixture in the presence of 8 mM MgCl 2 or 8 mM MnCl 2 in a reaction buffer containing increasing amounts of T7 DNA primase (0, 1.11, 3.33, and 10 µM). After incubation for 20 min at RT, the radioactive products were analyzed by electrophoresis through a 25% polyacrylamide gel containing 7 M urea and visualized using autoradiography. ( b ) Quantification of primase activity. The bands in the gel presented in a were analyzed using autoradiography.

    Article Snippet: Protein expression and purification All chemical reagents were molecular biology grade (Sigma); ATP, CTP, (Roche Molecular Biochemicals); Radio-labeled ATP or CTP (800 Ci/mmol) were from Perkin Elmer.

    Techniques: Sequencing, Incubation, Electrophoresis, Autoradiography, Activity Assay

    Dinucleotide synthesis by the T7 primase fragment catalyzed by Mn(II) or by Mg(II). ( a ) Experimental setup. ( b ) Single-turnover dinucleotide synthesis was initiated by mixing 25 µM T7 primase and 100 µM template (5′-GTCA 10 -3′) with 5 mM MnCl 2 , 1 nM γ - 32 P ATP and 1 mM CTP in a rapid quench-flow instrument. Reactions were quenched after various incubation times, and products were separated by gel electrophoresis. The fraction of dinucleotide product formed as a function of time was plotted for Mg(II) (green curve) and Mn(II) (red curve), and the curves were fit to a single-exponential function. ( c ) Dinucleotide synthesis was initiated by mixing (final concentrations) 10 µM T7 primase and 100 µM template with 10 mM MgCl 2 or 2 mM MnCl 2 , 1 mM ATP and 1 mM CTP (spiked with [α- 32 P CTP]) in a rapid quench-flow instrument. Reactions were quenched after various incubation times and products were separated by gel electrophoresis. The concentration of dinucleotide formed divided by the enzyme concentration is plotted versus time for MgCl 2 and MnCl 2 , and the curves were fit to either a linear or a burst equation. ( d ) Table listing the rate constants and the corresponding uncertainties that were used to fit the data.

    Journal: Scientific Reports

    Article Title: Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase

    doi: 10.1038/s41598-017-05534-3

    Figure Lengend Snippet: Dinucleotide synthesis by the T7 primase fragment catalyzed by Mn(II) or by Mg(II). ( a ) Experimental setup. ( b ) Single-turnover dinucleotide synthesis was initiated by mixing 25 µM T7 primase and 100 µM template (5′-GTCA 10 -3′) with 5 mM MnCl 2 , 1 nM γ - 32 P ATP and 1 mM CTP in a rapid quench-flow instrument. Reactions were quenched after various incubation times, and products were separated by gel electrophoresis. The fraction of dinucleotide product formed as a function of time was plotted for Mg(II) (green curve) and Mn(II) (red curve), and the curves were fit to a single-exponential function. ( c ) Dinucleotide synthesis was initiated by mixing (final concentrations) 10 µM T7 primase and 100 µM template with 10 mM MgCl 2 or 2 mM MnCl 2 , 1 mM ATP and 1 mM CTP (spiked with [α- 32 P CTP]) in a rapid quench-flow instrument. Reactions were quenched after various incubation times and products were separated by gel electrophoresis. The concentration of dinucleotide formed divided by the enzyme concentration is plotted versus time for MgCl 2 and MnCl 2 , and the curves were fit to either a linear or a burst equation. ( d ) Table listing the rate constants and the corresponding uncertainties that were used to fit the data.

    Article Snippet: Protein expression and purification All chemical reagents were molecular biology grade (Sigma); ATP, CTP, (Roche Molecular Biochemicals); Radio-labeled ATP or CTP (800 Ci/mmol) were from Perkin Elmer.

    Techniques: Flow Cytometry, Incubation, Nucleic Acid Electrophoresis, Concentration Assay

    HPLC profiles of T7 DNA primase reaction mixtures using DNA template CCCC GGGT CT 25 . Crude enzymatic mixtures were analyzed directly. Reaction conditions were as follows: T7 DNA primase reaction buffer, DTT and BSA as given in Material and Methods, 5 mM CTP, 1 mM GpppA (A) or 7Me GpppA (B) , 10 µM DNA template CCCC GGGT CT 25 (named dAC3) and 4 µM T7 DNA primase. Reactions were incubated at 37°C for 6 h. The first section (marked in gray) denotes the removal of proteic material and remaining CTP by an on-line cleaning procedure using a pre-column. The produced capped RNAs were then separated on the reverse-phase C18 column. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min, reaching 30% after 35 min and 50% after 40 min.

    Journal: Nucleic Acids Research

    Article Title: High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2?O positions

    doi: 10.1093/nar/gkl1119

    Figure Lengend Snippet: HPLC profiles of T7 DNA primase reaction mixtures using DNA template CCCC GGGT CT 25 . Crude enzymatic mixtures were analyzed directly. Reaction conditions were as follows: T7 DNA primase reaction buffer, DTT and BSA as given in Material and Methods, 5 mM CTP, 1 mM GpppA (A) or 7Me GpppA (B) , 10 µM DNA template CCCC GGGT CT 25 (named dAC3) and 4 µM T7 DNA primase. Reactions were incubated at 37°C for 6 h. The first section (marked in gray) denotes the removal of proteic material and remaining CTP by an on-line cleaning procedure using a pre-column. The produced capped RNAs were then separated on the reverse-phase C18 column. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min, reaching 30% after 35 min and 50% after 40 min.

    Article Snippet: Analytical scale Experiments were carried out in 20–40 µl reaction volume containing T7 DNA primase reaction buffer (40 mM Tris pH 7.5, 10 mM MgCl2 , 50 mM potassium glutamate, 1 μM ZnCl2 ), 10 mM DTT and 50 μg/ml BSA as well as CTP (Amersham Biosciences), cap analog (New England Biolabs), DNA oligonucleotides (Invitrogen, HPLC grade) and enzyme at concentrations given in table and figure legends.

    Techniques: High Performance Liquid Chromatography, Incubation, Produced

    Transfection of Cells with Liposomes for Induced Expression of Different Proteins (A) The schematic overview shows the process of mRNA incorporation in liposomes and subsequent transfection and mRNA translation. During transfection, the lipoplexes are endocytosed by the cells of interest. Inside the cell, the lipid layer is degraded and mRNA is released. In the cytosol, the mRNA is translated by ribosomes and the protein is subsequently released intra- or extracellularly. (B) The presence of Cy3-labeled EGFP-encoding mRNA after liposome uptake and expression of EGFP protein in cells 24 hr posttransfection.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Cationic Nanoliposomes Meet mRNA: Efficient Delivery of Modified mRNA Using Hemocompatible and Stable Vectors for Therapeutic Applications

    doi: 10.1016/j.omtn.2017.07.013

    Figure Lengend Snippet: Transfection of Cells with Liposomes for Induced Expression of Different Proteins (A) The schematic overview shows the process of mRNA incorporation in liposomes and subsequent transfection and mRNA translation. During transfection, the lipoplexes are endocytosed by the cells of interest. Inside the cell, the lipid layer is degraded and mRNA is released. In the cytosol, the mRNA is translated by ribosomes and the protein is subsequently released intra- or extracellularly. (B) The presence of Cy3-labeled EGFP-encoding mRNA after liposome uptake and expression of EGFP protein in cells 24 hr posttransfection.

    Article Snippet: For the fluorescent labeling of the mRNA, cy3-cytidine-triphosphate (PerkinElmer) was used.

    Techniques: Transfection, Expressing, Labeling

    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 P]CTP and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis *

    doi: 10.1074/jbc.M110.151316

    Figure Lengend Snippet: Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 P]CTP and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.

    Article Snippet: GTP was added at different concentrations before or at the same time as 0.5 m m ATP, 3′-dUTP, and 10 or 100 μ m CTP with 4 μCi of [α-32 P]CTP (3000 Ci·mmol−1 , PerkinElmer Life Sciences).

    Techniques: Incubation, TCA Precipitation

    H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 P-CTP for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.

    Journal: Nucleic Acids Research

    Article Title: Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome

    doi: 10.1093/nar/gkx724

    Figure Lengend Snippet: H 2 O 2 directly inhibits pausing by DSIF and NELF. ( A ) Elongation complexes were generated by first preincubating HeLa nuclear extract and immobilized template DNA for 30 min, and then pulsing with limiting α- 32 P-CTP for 30 s. Complexes were isolated by high salt wash and incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 . Addback mixtures containing buffer alone (Mock, lanes 1–4) or also containing 0.3 pmol DSIF and 0.6 pmol NELF (lanes 5–8) were also incubated 10 min with 1 mM DTT or indicated concentrations of H 2 O 2 prior to incubation with complexes for 5 min. Elongation rates were measured by chasing for 5 min with 500 μM cold ATP, UTP, GTP and CTP. 6% Urea–PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 50 min with 1 μM flavopiridol, then 10 min with 0.1% DMSO (flavopiridol only), 10 min with 50 μM PJ34, or 20 min with 50 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. DMSO was 0.2% in all conditions. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) Average normalized Pol II ChIP-Seq occupancies ±1.5 kb around 17592 EPDnew TSS. Suspension HeLa cells were treated 40 min with 1 μM flavopiridol, then 20 min with either 0.05% DMSO (flavopiridol only) or 20 μM PJ34. Cells were additionally treated 10 min with 0.3 mM H 2 O 2 as indicated. The vertical axis represents depth-adjusted signals that were background-subtracted and normalized using a window ±10 kb around genes.

    Article Snippet: Nuclear walk-on For each reaction, 1–3 × 105 nuclei were diluted to achieve the following reaction conditions ±1.33 μg/ml α-amanitin (Sigma A2263): 20 mM HEPES pH 7.6, 0.5% sarkosyl, 5 mM Mg(C2 H3 O2 )2 , 5 mM DTT, 150 mM KC2 H3 O2 , 0.25 U/μl SUPERase-In and 0.167 μM α-32 P-CTP (PerkinElmer BLU008H001MC).

    Techniques: Generated, Isolation, Incubation, Polyacrylamide Gel Electrophoresis, Chromatin Immunoprecipitation

    H 2 O 2 causes rapid accumulation, then creeping of promoter-proximal Pol II. ( A ) Capped nascent Pol II transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated with 0.3 mM H 2 O 2 for indicated times. No H 2 O 2 control profiles are duplicated in all panels. Capped transcripts were selected by treating Trizol-isolated RNAs with recombinant RNMT and SAM, and then using an immobilized anti-2,2,7-trimethylguanosine antibody. All plots in A were from the same gel (see Gel Appendix) and have the same vertical scale (relative signal). ( B ) The experiment in A, except all cells were treated 1 h with 1 μM flavopiridol prior to H 2 O 2 . All plots in B were from the same gel and have the same vertical scale (relative signal), which is reduced to match the paused Pol II peak height in A. ( C ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 1 h with 0.1% DMSO. 0.3 mM H 2 O 2 was added during the last 20 s, 1 min or 3 min of treatment as indicated ( n = 2). ( D ) The experiment in C, except all cells were treated 1 h with 1 μM flavopiridol instead of DMSO alone. DMSO was 0.1% in all conditions. Figure 2C and D were from the same gel and have the same vertical scale (relative signal). ( E ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 1 h with 1 μM flavopiridol. 0.3, 3 or 30 mM H 2 O 2 were added during the last 10 min of treatment as indicated. After a 5 min α- 32 P-CTP pulse, reactions were chased as indicated for 10 min with 500 μM cold ATP, UTP, GTP and CTP. All plots in Figure 2E were from the same gel and have the same vertical scale (relative signal).

    Journal: Nucleic Acids Research

    Article Title: Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome

    doi: 10.1093/nar/gkx724

    Figure Lengend Snippet: H 2 O 2 causes rapid accumulation, then creeping of promoter-proximal Pol II. ( A ) Capped nascent Pol II transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated with 0.3 mM H 2 O 2 for indicated times. No H 2 O 2 control profiles are duplicated in all panels. Capped transcripts were selected by treating Trizol-isolated RNAs with recombinant RNMT and SAM, and then using an immobilized anti-2,2,7-trimethylguanosine antibody. All plots in A were from the same gel (see Gel Appendix) and have the same vertical scale (relative signal). ( B ) The experiment in A, except all cells were treated 1 h with 1 μM flavopiridol prior to H 2 O 2 . All plots in B were from the same gel and have the same vertical scale (relative signal), which is reduced to match the paused Pol II peak height in A. ( C ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 1 h with 0.1% DMSO. 0.3 mM H 2 O 2 was added during the last 20 s, 1 min or 3 min of treatment as indicated ( n = 2). ( D ) The experiment in C, except all cells were treated 1 h with 1 μM flavopiridol instead of DMSO alone. DMSO was 0.1% in all conditions. Figure 2C and D were from the same gel and have the same vertical scale (relative signal). ( E ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 1 h with 1 μM flavopiridol. 0.3, 3 or 30 mM H 2 O 2 were added during the last 10 min of treatment as indicated. After a 5 min α- 32 P-CTP pulse, reactions were chased as indicated for 10 min with 500 μM cold ATP, UTP, GTP and CTP. All plots in Figure 2E were from the same gel and have the same vertical scale (relative signal).

    Article Snippet: Nuclear walk-on For each reaction, 1–3 × 105 nuclei were diluted to achieve the following reaction conditions ±1.33 μg/ml α-amanitin (Sigma A2263): 20 mM HEPES pH 7.6, 0.5% sarkosyl, 5 mM Mg(C2 H3 O2 )2 , 5 mM DTT, 150 mM KC2 H3 O2 , 0.25 U/μl SUPERase-In and 0.167 μM α-32 P-CTP (PerkinElmer BLU008H001MC).

    Techniques: Isolation, Recombinant

    The nuclear walk-on assay quantitatively and precisely measures engaged Pol II. ( A ) Steps in the nuclear walk-on assay are illustrated. ( B ) Adherent HeLa cells were transfected 48 h before isolation of nuclei with 1.5 nM NELF-A siRNA (NELF KD) or lipid reagent only (Mock). Cells were also treated 1 h before isolation with 0.1% DMSO or 1 μM flavopiridol (Flavo). Nuclear walk-ons were performed using a 6 min α- 32 P-CTP pulse in the absence or presence of 2 μg/ml α-amanitin (α-aman). After 6% Urea-PAGE, ethidium bromide was used to visualize nuclear RNAs. A representative gel from nuclear walk-ons performed in triplicate is shown here. ( C ) Phosphorimage to visualize radiolabeled nascent transcripts from B. ( D ) Average amanitin-sensitive Pol II nascent transcript profiles from triplicate nuclear walk-ons as represented in B and C. For each replicate, signals from each lane in the phosphorimage were normalized using quantifications of cold nuclear RNAs in the ethidium bromide stain. Then, α-amanitin-insensitive signals were subtracted from total signals. Triplicate profiles (see Supplementary Figure S1 ) were then averaged as described in the Materials and Methods. Inset: western blot of NELF-A; a nonspecific band (n.s.) indicates even loading. ( E ) PRO-Seq paired-end reads over the DDIT4 gene and upstream enhancer regions. Adherent HeLa cells were treated 1 h with either 0.1% DMSO or 1 μM flavopiridol. ( F ) Plot of sense or divergent PRO-Seq fragments lengths near Pol II promoters.

    Journal: Nucleic Acids Research

    Article Title: Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome

    doi: 10.1093/nar/gkx724

    Figure Lengend Snippet: The nuclear walk-on assay quantitatively and precisely measures engaged Pol II. ( A ) Steps in the nuclear walk-on assay are illustrated. ( B ) Adherent HeLa cells were transfected 48 h before isolation of nuclei with 1.5 nM NELF-A siRNA (NELF KD) or lipid reagent only (Mock). Cells were also treated 1 h before isolation with 0.1% DMSO or 1 μM flavopiridol (Flavo). Nuclear walk-ons were performed using a 6 min α- 32 P-CTP pulse in the absence or presence of 2 μg/ml α-amanitin (α-aman). After 6% Urea-PAGE, ethidium bromide was used to visualize nuclear RNAs. A representative gel from nuclear walk-ons performed in triplicate is shown here. ( C ) Phosphorimage to visualize radiolabeled nascent transcripts from B. ( D ) Average amanitin-sensitive Pol II nascent transcript profiles from triplicate nuclear walk-ons as represented in B and C. For each replicate, signals from each lane in the phosphorimage were normalized using quantifications of cold nuclear RNAs in the ethidium bromide stain. Then, α-amanitin-insensitive signals were subtracted from total signals. Triplicate profiles (see Supplementary Figure S1 ) were then averaged as described in the Materials and Methods. Inset: western blot of NELF-A; a nonspecific band (n.s.) indicates even loading. ( E ) PRO-Seq paired-end reads over the DDIT4 gene and upstream enhancer regions. Adherent HeLa cells were treated 1 h with either 0.1% DMSO or 1 μM flavopiridol. ( F ) Plot of sense or divergent PRO-Seq fragments lengths near Pol II promoters.

    Article Snippet: Nuclear walk-on For each reaction, 1–3 × 105 nuclei were diluted to achieve the following reaction conditions ±1.33 μg/ml α-amanitin (Sigma A2263): 20 mM HEPES pH 7.6, 0.5% sarkosyl, 5 mM Mg(C2 H3 O2 )2 , 5 mM DTT, 150 mM KC2 H3 O2 , 0.25 U/μl SUPERase-In and 0.167 μM α-32 P-CTP (PerkinElmer BLU008H001MC).

    Techniques: Transfection, Isolation, Polyacrylamide Gel Electrophoresis, Staining, Western Blot

    H 2 O 2 inhibits Pol II termination in vitro and increases Pol II engagement in cells. ( A ) Preinitiation complexes were formed on immobilized template DNA in the presence of indicated concentrations of H 2 O 2 . Elongation complexes were pulsed 30 s with limiting α- 32 P-CTP and chased as indicated for 3 min with 500 μM cold ATP, UTP, GTP and CTP. Lengths of limiting-CTP transcripts are indicated; brackets indicate sizes of capped and uncapped transcripts. 6% Urea-PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 100 min with 1 μM flavopiridol, and then 3 min with 0.3 mM H 2 O 2 as indicated. 500 nM triptolide was added either during the last 30 min of flavopiridol treatment, or during all 100 min of flavopiridol treatment. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) 0.1, 1 or 10 μM triptolide was added to preinitiation complexes 30, 10, 3 or 1 min before a 30 s limiting α- 32 P-CTP pulse. Labeled non-tRNA transcripts were quantified, adjusted for loading using cold nuclear RNAs, and normalized to reactions lacking triptolide. Error bars represent S.E.M. from three replicates. ( D ) Preinitiation complexes were formed on immobilized template DNA in the presence of 1 μM flavopiridol alone (C; control) or in combination with 0.3 mM H 2 O 2 (H). Elongation complexes were pulsed 30 s with limiting α- 32 P-CTP and chased 10 min with 500 μM cold ATP, UTP, GTP and CTP. Labeled transcripts from bead-bound (B) and supernatant (S) fractions were quantified to measure termination. Runoff transcripts (508 nt) were considered bound and tRNAs were excluded. Error bars represent S.E.M. and p -values are from 3 replicates (see Gel Appendix). 12% Urea–PAGE. ( E ) Top panel: western blot of RPB1 (sc-55492) and Ponceau S staining. Total (T) or cytosolic (C) and nuclear (N) fractions were obtained from adherent HeLa cells treated 1 h with 0.1% DMSO or 1 μM flavopiridol, and then 10 min with 0.3 mM H 2 O 2 as indicated. Shown are two representative blots for RBP1. The bottom plot pairs with the Ponceau S staining. 4–20% SDS-PAGE. Bottom panel: plot of the percentage of RPB1 in the cytosolic (C/C+N) or nuclear (N/C+N) fractions from five replicates.

    Journal: Nucleic Acids Research

    Article Title: Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome

    doi: 10.1093/nar/gkx724

    Figure Lengend Snippet: H 2 O 2 inhibits Pol II termination in vitro and increases Pol II engagement in cells. ( A ) Preinitiation complexes were formed on immobilized template DNA in the presence of indicated concentrations of H 2 O 2 . Elongation complexes were pulsed 30 s with limiting α- 32 P-CTP and chased as indicated for 3 min with 500 μM cold ATP, UTP, GTP and CTP. Lengths of limiting-CTP transcripts are indicated; brackets indicate sizes of capped and uncapped transcripts. 6% Urea-PAGE. ( B ) Amanitin-sensitive Pol II nascent transcript profiles from a nuclear walk-on using nuclei from adherent HeLa cells treated 100 min with 1 μM flavopiridol, and then 3 min with 0.3 mM H 2 O 2 as indicated. 500 nM triptolide was added either during the last 30 min of flavopiridol treatment, or during all 100 min of flavopiridol treatment. The vertical axis represents relative signal from Pol II and all curves were from the same gel. ( C ) 0.1, 1 or 10 μM triptolide was added to preinitiation complexes 30, 10, 3 or 1 min before a 30 s limiting α- 32 P-CTP pulse. Labeled non-tRNA transcripts were quantified, adjusted for loading using cold nuclear RNAs, and normalized to reactions lacking triptolide. Error bars represent S.E.M. from three replicates. ( D ) Preinitiation complexes were formed on immobilized template DNA in the presence of 1 μM flavopiridol alone (C; control) or in combination with 0.3 mM H 2 O 2 (H). Elongation complexes were pulsed 30 s with limiting α- 32 P-CTP and chased 10 min with 500 μM cold ATP, UTP, GTP and CTP. Labeled transcripts from bead-bound (B) and supernatant (S) fractions were quantified to measure termination. Runoff transcripts (508 nt) were considered bound and tRNAs were excluded. Error bars represent S.E.M. and p -values are from 3 replicates (see Gel Appendix). 12% Urea–PAGE. ( E ) Top panel: western blot of RPB1 (sc-55492) and Ponceau S staining. Total (T) or cytosolic (C) and nuclear (N) fractions were obtained from adherent HeLa cells treated 1 h with 0.1% DMSO or 1 μM flavopiridol, and then 10 min with 0.3 mM H 2 O 2 as indicated. Shown are two representative blots for RBP1. The bottom plot pairs with the Ponceau S staining. 4–20% SDS-PAGE. Bottom panel: plot of the percentage of RPB1 in the cytosolic (C/C+N) or nuclear (N/C+N) fractions from five replicates.

    Article Snippet: Nuclear walk-on For each reaction, 1–3 × 105 nuclei were diluted to achieve the following reaction conditions ±1.33 μg/ml α-amanitin (Sigma A2263): 20 mM HEPES pH 7.6, 0.5% sarkosyl, 5 mM Mg(C2 H3 O2 )2 , 5 mM DTT, 150 mM KC2 H3 O2 , 0.25 U/μl SUPERase-In and 0.167 μM α-32 P-CTP (PerkinElmer BLU008H001MC).

    Techniques: In Vitro, Polyacrylamide Gel Electrophoresis, Labeling, Western Blot, Staining, SDS Page