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  • 95
    Jena Bioscience utp
    Effect of NDPs on NS5BΔ21 RNA polymerase activity. (A) Polyacrylamide gel showing the products for de novo (DN), primer extension (PE) and template switching (TS) obtained with HCV NS5BΔ21 at increasing concentrations (0, 166, 333, 500, 800, and 1000 μM) of UDP in the presence of ATP and <t>UTP</t> at a final concentration of 100 μM, GTP at 500 μM, and radiolabeled α 32 <t>P-CTP</t> (left panel). Graphic representation of densitometric values obtained from the electropherogram shown in A (red diamonds, yellow triangles, and black squares correspond to de novo (DN), primer extension (PE), and template switching (TS) activities, respectively) (right panel). (B) Corresponds to experiments as in A but for increasing concentrations of ADP. (C) corresponds to experiments as in A but for increasing concentrations of GDP. Activities were normalized to their maximum values. Densitometric data represent the mean of at least three independent experiments. Error bars correspond to standard error of the mean. Horizontal lines indicate statistically significant differences (Student’s T-test) between activity values, using the same color code as the activity type. Details of the activity measurements are given in Materials and Methods. Significance (Student’s T-test): ** p
    Utp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher utp
    Analysis of the 5′ terminal groups of the product RNAs. Transcription was performed in the absence of <t>UTP</t> to stall the polymerase at position 14 of the template. The reaction products were recovered by phenol extraction and ethanol precipitation, resuspended in the appropriate buffer and incubated in the presence (+) or absence (–) of CIP, TAP or GTase as indicated. The reaction products were then analysed by denaturing electrophoresis using 7 M urea 18% PAGE. The bands corresponding to the major 13 nt unprimed product and mRNA primed products (*) are indicated. ( A ) Lanes 1–4, transcription in the absence of any added primer. Lanes 5–8, <t>ApG</t> primed transcription. Lanes 9–12, unprimed transcription in the presence of 0.4 mM m7 GpppG. Lanes 13–16, globin mRNA primed transcription. ( B ) Product RNAs from ApG (lanes 1 and 2) or unprimed reactions (lanes 3 and 4) were prepared as above and depleted of template RNA as described in the Materials and Methods. The samples were then treated with GTase (lanes 2 and 4) or mock treated (lanes 1 and 3) and analysed by PAGE as above.
    Utp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1065 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore utp
    Chronic esomeprazole does not disrupt epithelial integrity of CF hAECs. (A) Effect of increasing concentrations of apical esomeprazole on TransEpithelial Electrical Resistance (TEER) and Fluorescein Flux measured in Ussing chambers in the presence of a basolateral to apical Cl - gradient ( n = 5, five donors). (B,C) Effect of increasing concentrations of apical eso on resting short-circuit current (Isc, B , n = 5), and (C) <t>amiloride-sensitive</t> Isc (ΔIsc (amil) , black triangle, n = 5), Fsk-induced Isc (ΔIsc (Fsk) , open triangle, n = 5), CFTRinh172-sensitive Isc (ΔIsc (172) , open diamond, n = 5) and <t>UTP-induced</t> Isc (ΔIsc (UTP) , black circles, n = 5).
    Utp, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare α 32 p utp
    Effect of cre (2C)mut1 on VPg uridylylation in reconstituted reactions. VPg uridylylation was measured in reconstituted reaction mixtures containing 3D pol , VPg, [α- 32 <t>P]UTP,</t> the indicated RNAs, and 3CD as described in Materials and Methods (lanes 2 to 5). 32 P-labeled VPgpUpU synthesized in these reactions was resolved by SDS-PAGE (9 to 18% polyacrylamide). [ 35 S]methionine-labeled poliovirus proteins were used as markers and are shown in lane 1.
    α 32 P Utp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 1907 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Boehringer Mannheim digoxigenin uridine triphosphate
    Demonstration of CENP-E messenger RNA and protein in rheumatoid synovium by in situ hybridization and immunohistochemistry. (a, b) A <t>digoxigenin-labelled</t> RNA probe transcribed from the amplified RNA arbitrarily primed-PCR gene product is used on rheumatoid arthritis snap-frozen sections. Double-labelling for CENP-E messenger RNA (black staining), and alkaline phosphatase antialkaline phosphatase counterstaining using anti-fibroblast antibodies (red staining) shows CENP-E expression in numerous fibroblasts througout the synovium (a, arrows). (b) The intensive CENP-E messenger RNA expression in two synovial fibroblasts. (c) Shows numerous fibroblasts expressing CENP-E protein [black staining (arrows), counterstaining with fast red]. Original magnifications × 300 (a, c) and × 600 (b).
    Digoxigenin Uridine Triphosphate, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours α 32 p utp
    In vitro RdRp assay with exogenous RNA templates. Approximately 50 ng of RNA template r138/40A (the 3′ UTR of Bamboo mosaic virus (BaMV)) in (a) and Ba-77 (the 3′-end 77 nts of BaMV minus-strand) in (b) were incubated with BaMV RdRp complex for the in vitro RNA synthesis in the presence of different concentrations (0–100 mM) of glutathione (GSH) as indicated. The RdRp products labeled with [α- 32 P] <t>UTP</t> as indicated bands were separated on a 5% acrylamide gel and quantified by a phosphorimager. (c) The relative RdRp template activities were plotted according to the data derived from (a) and (b). The banding density of the in vitro RdRp assay with either r138/40A or Ba-77 was set as 100% in the absence of GSH (0 mM). Each spot on the plot was the average ± SE of at least three independent experiments.
    α 32 P Utp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim digoxigenin 11 utp
    Expression pattern of the zen ( A and B ) and twist ( C and D ) genes in blastoderm embryos. Expression is visualised by whole mount in situ hybridisation using a <t>digoxigenin-11-UTP-labelled</t> antisense RNA probe followed by immunological staining. Embryos are oriented with anterior to the left and dorsal up (A and B) or in ventral view (C and D). (A) and (C), wild-type embryos; (B) and (D), DSP1 mutant embryos.
    Digoxigenin 11 Utp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PerkinElmer biotin 11 utp
    Expression pattern of the zen ( A and B ) and twist ( C and D ) genes in blastoderm embryos. Expression is visualised by whole mount in situ hybridisation using a <t>digoxigenin-11-UTP-labelled</t> antisense RNA probe followed by immunological staining. Embryos are oriented with anterior to the left and dorsal up (A and B) or in ventral view (C and D). (A) and (C), wild-type embryos; (B) and (D), DSP1 mutant embryos.
    Biotin 11 Utp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore uridine 5 triphosphate utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    Uridine 5 Triphosphate Utp, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem biotin 16 utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    Biotin 16 Utp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Enzo Biochem biotinylated utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    Biotinylated Utp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer dnp 11 utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    Dnp 11 Utp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Boehringer Mannheim digoxigenin labeled utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    Digoxigenin Labeled Utp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher 5 3 aminoallyl utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    5 3 Aminoallyl Utp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher aminoallyl utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    Aminoallyl Utp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher bio 16 utp
    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate <t>(UTP)</t> in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.
    Bio 16 Utp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare cy3 utp
    aRNA yield after different incubation times. 5 μg of total RNA were amplified and labeled (either <t>Cy3-UTP</t> or Cy5-UTP) or non-labeled ('no Dye' control) with the T7-System for 2, 4, 6, 8 and 16 hours (n = 3). The aRNA was purified by column chromatography and quantified by UV-spectrophotometry.
    Cy3 Utp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant α 32 p utp
    Affinity-purified ytmQ gene product (BsTrmB) catalyzes the formation of m 7 G in T7 transcripts of B.subtilis tRNA Phe . ( A ) B.subtilis tRNA Phe (5 µg) was incubated with 5 µg of purified BsTrmB protein and 15 µM of [methyl- 14 C]AdoMet (58 mCi/mmol) in TM buffer (50 mM Tris–HCl and 10 mM MgCl 2 , pH8) in a total volume of 200 µl. After 30 min incubation at 37°C the tRNA was recovered and digested with nuclease P1. The resulting nucleotides were analyzed by 2D-TLC and autoradiography as described ( 42 ). First dimension developed with isobutyric acid/conc. NH 4 OH/water (66:1:33; v/v/v); second dimension developed with conc. HCl/isopropanol/water (17.6:68:14.4; v/v/v). ( B ) [α- 32 <t>P]UTP-labeled</t> in vitro transcribed B.subtilis tRNA Phe (5 × 10 5 c.p.m.) was incubated with 5 µg of purified BsTrmB and 30 µM AdoMet in TM buffer in a total volume of 300 µl. After 30 min incubation at 37°C, the tRNA was recovered, hydrolyzed by RNAse T2 and the resulting nucleotides were analyzed as above.
    α 32 P Utp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem biotin utp
    Affinity-purified ytmQ gene product (BsTrmB) catalyzes the formation of m 7 G in T7 transcripts of B.subtilis tRNA Phe . ( A ) B.subtilis tRNA Phe (5 µg) was incubated with 5 µg of purified BsTrmB protein and 15 µM of [methyl- 14 C]AdoMet (58 mCi/mmol) in TM buffer (50 mM Tris–HCl and 10 mM MgCl 2 , pH8) in a total volume of 200 µl. After 30 min incubation at 37°C the tRNA was recovered and digested with nuclease P1. The resulting nucleotides were analyzed by 2D-TLC and autoradiography as described ( 42 ). First dimension developed with isobutyric acid/conc. NH 4 OH/water (66:1:33; v/v/v); second dimension developed with conc. HCl/isopropanol/water (17.6:68:14.4; v/v/v). ( B ) [α- 32 <t>P]UTP-labeled</t> in vitro transcribed B.subtilis tRNA Phe (5 × 10 5 c.p.m.) was incubated with 5 µg of purified BsTrmB and 30 µM AdoMet in TM buffer in a total volume of 300 µl. After 30 min incubation at 37°C, the tRNA was recovered, hydrolyzed by RNAse T2 and the resulting nucleotides were analyzed as above.
    Biotin Utp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DuPont de Nemours s utp
    Affinity-purified ytmQ gene product (BsTrmB) catalyzes the formation of m 7 G in T7 transcripts of B.subtilis tRNA Phe . ( A ) B.subtilis tRNA Phe (5 µg) was incubated with 5 µg of purified BsTrmB protein and 15 µM of [methyl- 14 C]AdoMet (58 mCi/mmol) in TM buffer (50 mM Tris–HCl and 10 mM MgCl 2 , pH8) in a total volume of 200 µl. After 30 min incubation at 37°C the tRNA was recovered and digested with nuclease P1. The resulting nucleotides were analyzed by 2D-TLC and autoradiography as described ( 42 ). First dimension developed with isobutyric acid/conc. NH 4 OH/water (66:1:33; v/v/v); second dimension developed with conc. HCl/isopropanol/water (17.6:68:14.4; v/v/v). ( B ) [α- 32 <t>P]UTP-labeled</t> in vitro transcribed B.subtilis tRNA Phe (5 × 10 5 c.p.m.) was incubated with 5 µg of purified BsTrmB and 30 µM AdoMet in TM buffer in a total volume of 300 µl. After 30 min incubation at 37°C, the tRNA was recovered, hydrolyzed by RNAse T2 and the resulting nucleotides were analyzed as above.
    S Utp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare cy5 utp
    Co-localization of endogenous Staufen protein and injected VLE RNA. Stage II oocytes were injected with 500 nM <t>Cy5-UTP-labeled</t> VLE ) at 1:250 dilution and an Alexa-546-secondary antibody (Thermo-Fisher) at 1:500 dilution. Oocytes were imaged on a Zeiss LSM 510 Meta Confocal Laser Scanning Microscope using a 20× objective. Shown is a confocal section with (A) VLE RNA in red, (B) Staufen protein in green, and (C) merged red and green channels, showing Staufen protein and VLE RNA colocalization. The oocyte is oriented with the vegetal pole at the bottom and the scale bars = 50 µm.
    Cy5 Utp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of NDPs on NS5BΔ21 RNA polymerase activity. (A) Polyacrylamide gel showing the products for de novo (DN), primer extension (PE) and template switching (TS) obtained with HCV NS5BΔ21 at increasing concentrations (0, 166, 333, 500, 800, and 1000 μM) of UDP in the presence of ATP and UTP at a final concentration of 100 μM, GTP at 500 μM, and radiolabeled α 32 P-CTP (left panel). Graphic representation of densitometric values obtained from the electropherogram shown in A (red diamonds, yellow triangles, and black squares correspond to de novo (DN), primer extension (PE), and template switching (TS) activities, respectively) (right panel). (B) Corresponds to experiments as in A but for increasing concentrations of ADP. (C) corresponds to experiments as in A but for increasing concentrations of GDP. Activities were normalized to their maximum values. Densitometric data represent the mean of at least three independent experiments. Error bars correspond to standard error of the mean. Horizontal lines indicate statistically significant differences (Student’s T-test) between activity values, using the same color code as the activity type. Details of the activity measurements are given in Materials and Methods. Significance (Student’s T-test): ** p

    Journal: bioRxiv

    Article Title: Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

    doi: 10.1101/2020.02.21.959536

    Figure Lengend Snippet: Effect of NDPs on NS5BΔ21 RNA polymerase activity. (A) Polyacrylamide gel showing the products for de novo (DN), primer extension (PE) and template switching (TS) obtained with HCV NS5BΔ21 at increasing concentrations (0, 166, 333, 500, 800, and 1000 μM) of UDP in the presence of ATP and UTP at a final concentration of 100 μM, GTP at 500 μM, and radiolabeled α 32 P-CTP (left panel). Graphic representation of densitometric values obtained from the electropherogram shown in A (red diamonds, yellow triangles, and black squares correspond to de novo (DN), primer extension (PE), and template switching (TS) activities, respectively) (right panel). (B) Corresponds to experiments as in A but for increasing concentrations of ADP. (C) corresponds to experiments as in A but for increasing concentrations of GDP. Activities were normalized to their maximum values. Densitometric data represent the mean of at least three independent experiments. Error bars correspond to standard error of the mean. Horizontal lines indicate statistically significant differences (Student’s T-test) between activity values, using the same color code as the activity type. Details of the activity measurements are given in Materials and Methods. Significance (Student’s T-test): ** p

    Article Snippet: To this end, 50 µl of 20 pmol/µl UTP, CTP, ATP and GTP (Jena Bioscience), were separated prior to sample analysis.

    Techniques: Activity Assay, Concentration Assay

    Effect of Gua on NS5BΔ21 RNA polymerase activity. (A) Recombinant HCV NS5B Δ 21 polymerase was added to a reaction containing a 540-nt RNA template [ 18 ], the four nucleoside-triphosphates (ATP, CTP, GTP, and UTP) and the indicated concentrations of Gua. Product quantification from three replicates (average ± SEM) and a representative experiment (below) are shown. Polymerase activity is normalized with respect to its maximum activity. The band indicates a new synthesis RNA product of 540 nt length. (B) A representative experiment as in A, but using the 19-nt LE19 RNA as a template. DN, PE, and TS indicate reaction products of de novo synthesis, primer extension, and template switching, respectively [ 54 ]. Procedures are detailed in Materials and Methods. Significance (Student’s T-test): ** p

    Journal: bioRxiv

    Article Title: Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

    doi: 10.1101/2020.02.21.959536

    Figure Lengend Snippet: Effect of Gua on NS5BΔ21 RNA polymerase activity. (A) Recombinant HCV NS5B Δ 21 polymerase was added to a reaction containing a 540-nt RNA template [ 18 ], the four nucleoside-triphosphates (ATP, CTP, GTP, and UTP) and the indicated concentrations of Gua. Product quantification from three replicates (average ± SEM) and a representative experiment (below) are shown. Polymerase activity is normalized with respect to its maximum activity. The band indicates a new synthesis RNA product of 540 nt length. (B) A representative experiment as in A, but using the 19-nt LE19 RNA as a template. DN, PE, and TS indicate reaction products of de novo synthesis, primer extension, and template switching, respectively [ 54 ]. Procedures are detailed in Materials and Methods. Significance (Student’s T-test): ** p

    Article Snippet: To this end, 50 µl of 20 pmol/µl UTP, CTP, ATP and GTP (Jena Bioscience), were separated prior to sample analysis.

    Techniques: Activity Assay, Recombinant

    Effect of nucleoside-triphosphate concentration on NS5BΔ21 RNA polymerase activity. ( A) Polyacrylamide gel showing the products for de novo (DN), primer extension (PE) and template switching (TS) obtained with HCV NS5BΔ21 at increasing concentrations (100, 500, 800, and 1000 μM) of UTP in the presence of radiolabeled α 32 P-CTP. ATP and GTP concentrations were maintained at 100 μM and 500 μM, respectively (left panel). Graphic representation of densitometric values obtained from the electropherogram shown in A (red diamonds, yellow triangles, and black squares correspond to de novo (DN), primer extension (PE), and template switching (TS) activities, respectively) (right panel). (B) Corresponds to experiments as in A but for increasing concentrations of ATP, with UTP and GTP maintained at 100 μM and 500 μM, respectively. (C) Corresponds to experiments as in A but for increasing concentrations of GTP, with ATP and UTP both maintained at 100 μM. Activities were normalized to their maximum values. Densitometric data represent the mean of at least three independent experiments. Error bars correspond to standard error of the mean. Horizontal lines indicate statistically significant differences (Student’s T-test) between the activity values that link, using the same color code as the activity type. Details of the activity measurements are given in Materials and Methods. Significance (Student’s T-test): *** p

    Journal: bioRxiv

    Article Title: Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

    doi: 10.1101/2020.02.21.959536

    Figure Lengend Snippet: Effect of nucleoside-triphosphate concentration on NS5BΔ21 RNA polymerase activity. ( A) Polyacrylamide gel showing the products for de novo (DN), primer extension (PE) and template switching (TS) obtained with HCV NS5BΔ21 at increasing concentrations (100, 500, 800, and 1000 μM) of UTP in the presence of radiolabeled α 32 P-CTP. ATP and GTP concentrations were maintained at 100 μM and 500 μM, respectively (left panel). Graphic representation of densitometric values obtained from the electropherogram shown in A (red diamonds, yellow triangles, and black squares correspond to de novo (DN), primer extension (PE), and template switching (TS) activities, respectively) (right panel). (B) Corresponds to experiments as in A but for increasing concentrations of ATP, with UTP and GTP maintained at 100 μM and 500 μM, respectively. (C) Corresponds to experiments as in A but for increasing concentrations of GTP, with ATP and UTP both maintained at 100 μM. Activities were normalized to their maximum values. Densitometric data represent the mean of at least three independent experiments. Error bars correspond to standard error of the mean. Horizontal lines indicate statistically significant differences (Student’s T-test) between the activity values that link, using the same color code as the activity type. Details of the activity measurements are given in Materials and Methods. Significance (Student’s T-test): *** p

    Article Snippet: To this end, 50 µl of 20 pmol/µl UTP, CTP, ATP and GTP (Jena Bioscience), were separated prior to sample analysis.

    Techniques: Concentration Assay, Activity Assay

    Analysis of the 5′ terminal groups of the product RNAs. Transcription was performed in the absence of UTP to stall the polymerase at position 14 of the template. The reaction products were recovered by phenol extraction and ethanol precipitation, resuspended in the appropriate buffer and incubated in the presence (+) or absence (–) of CIP, TAP or GTase as indicated. The reaction products were then analysed by denaturing electrophoresis using 7 M urea 18% PAGE. The bands corresponding to the major 13 nt unprimed product and mRNA primed products (*) are indicated. ( A ) Lanes 1–4, transcription in the absence of any added primer. Lanes 5–8, ApG primed transcription. Lanes 9–12, unprimed transcription in the presence of 0.4 mM m7 GpppG. Lanes 13–16, globin mRNA primed transcription. ( B ) Product RNAs from ApG (lanes 1 and 2) or unprimed reactions (lanes 3 and 4) were prepared as above and depleted of template RNA as described in the Materials and Methods. The samples were then treated with GTase (lanes 2 and 4) or mock treated (lanes 1 and 3) and analysed by PAGE as above.

    Journal: Nucleic Acids Research

    Article Title: Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase

    doi:

    Figure Lengend Snippet: Analysis of the 5′ terminal groups of the product RNAs. Transcription was performed in the absence of UTP to stall the polymerase at position 14 of the template. The reaction products were recovered by phenol extraction and ethanol precipitation, resuspended in the appropriate buffer and incubated in the presence (+) or absence (–) of CIP, TAP or GTase as indicated. The reaction products were then analysed by denaturing electrophoresis using 7 M urea 18% PAGE. The bands corresponding to the major 13 nt unprimed product and mRNA primed products (*) are indicated. ( A ) Lanes 1–4, transcription in the absence of any added primer. Lanes 5–8, ApG primed transcription. Lanes 9–12, unprimed transcription in the presence of 0.4 mM m7 GpppG. Lanes 13–16, globin mRNA primed transcription. ( B ) Product RNAs from ApG (lanes 1 and 2) or unprimed reactions (lanes 3 and 4) were prepared as above and depleted of template RNA as described in the Materials and Methods. The samples were then treated with GTase (lanes 2 and 4) or mock treated (lanes 1 and 3) and analysed by PAGE as above.

    Article Snippet: Typical reaction conditions were 50 mM Tris pH 8.2, 100 mM KCl, 2 mM MgCl2 , 100 ng tRNA, 10 mM DTT, 10 pmol 3′V template, 5 µCi [α-32 P]GTP (400 Ci/mmol; Amersham Pharmacia Biotech Ltd), 0.8 mM ATP, 0.4 mM CTP, 0.2 mM UTP, 1 µM GTP and 0.5 mM ApG or 20 ng globin mRNA (Gibco-BRL).

    Techniques: Ethanol Precipitation, Incubation, Electrophoresis, Polyacrylamide Gel Electrophoresis

    Chronic esomeprazole does not disrupt epithelial integrity of CF hAECs. (A) Effect of increasing concentrations of apical esomeprazole on TransEpithelial Electrical Resistance (TEER) and Fluorescein Flux measured in Ussing chambers in the presence of a basolateral to apical Cl - gradient ( n = 5, five donors). (B,C) Effect of increasing concentrations of apical eso on resting short-circuit current (Isc, B , n = 5), and (C) amiloride-sensitive Isc (ΔIsc (amil) , black triangle, n = 5), Fsk-induced Isc (ΔIsc (Fsk) , open triangle, n = 5), CFTRinh172-sensitive Isc (ΔIsc (172) , open diamond, n = 5) and UTP-induced Isc (ΔIsc (UTP) , black circles, n = 5).

    Journal: Frontiers in Pharmacology

    Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells

    doi: 10.3389/fphar.2018.01462

    Figure Lengend Snippet: Chronic esomeprazole does not disrupt epithelial integrity of CF hAECs. (A) Effect of increasing concentrations of apical esomeprazole on TransEpithelial Electrical Resistance (TEER) and Fluorescein Flux measured in Ussing chambers in the presence of a basolateral to apical Cl - gradient ( n = 5, five donors). (B,C) Effect of increasing concentrations of apical eso on resting short-circuit current (Isc, B , n = 5), and (C) amiloride-sensitive Isc (ΔIsc (amil) , black triangle, n = 5), Fsk-induced Isc (ΔIsc (Fsk) , open triangle, n = 5), CFTRinh172-sensitive Isc (ΔIsc (172) , open diamond, n = 5) and UTP-induced Isc (ΔIsc (UTP) , black circles, n = 5).

    Article Snippet: Ouabain (O3125), esomeprazole (E7906), N -Acetyl- L -cysteine (A7250), amiloride (A7410), fluorescein (F6377), mitomycin C (M0503), and UTP (U6750) were purchased from Sigma-Aldrich.

    Techniques:

    Effect of cre (2C)mut1 on VPg uridylylation in reconstituted reactions. VPg uridylylation was measured in reconstituted reaction mixtures containing 3D pol , VPg, [α- 32 P]UTP, the indicated RNAs, and 3CD as described in Materials and Methods (lanes 2 to 5). 32 P-labeled VPgpUpU synthesized in these reactions was resolved by SDS-PAGE (9 to 18% polyacrylamide). [ 35 S]methionine-labeled poliovirus proteins were used as markers and are shown in lane 1.

    Journal: Journal of Virology

    Article Title: Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

    doi: 10.1128/JVI.77.9.5136-5144.2003

    Figure Lengend Snippet: Effect of cre (2C)mut1 on VPg uridylylation in reconstituted reactions. VPg uridylylation was measured in reconstituted reaction mixtures containing 3D pol , VPg, [α- 32 P]UTP, the indicated RNAs, and 3CD as described in Materials and Methods (lanes 2 to 5). 32 P-labeled VPgpUpU synthesized in these reactions was resolved by SDS-PAGE (9 to 18% polyacrylamide). [ 35 S]methionine-labeled poliovirus proteins were used as markers and are shown in lane 1.

    Article Snippet: The uridylylation reactions were identical to those described above for the RNA replication assays, except the reaction mixtures contained 5 μM UTP, which was provided by the addition of 100 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham) and 250 μM (each) ATP, CTP, and GTP.

    Techniques: Labeling, Synthesized, SDS Page

    Synthesis of uridylylated VPg in PIRCs. HeLa S10 translation-RNA replication reaction mixtures containing 2 mM guanidine HCl were prepared with PV1 RNA and PV1VPgY3F RNA. VPg uridylylation was measured in PIRCs resuspended in reaction mixtures containing [α- 32 P]UTP. The labeled VPgpUpU synthesized in these reactions was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods and resolved by SDS-PAGE. [ 35 S]methionine-labeled poliovirus proteins were used as markers (lane 7).

    Journal: Journal of Virology

    Article Title: Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

    doi: 10.1128/JVI.77.9.5136-5144.2003

    Figure Lengend Snippet: Synthesis of uridylylated VPg in PIRCs. HeLa S10 translation-RNA replication reaction mixtures containing 2 mM guanidine HCl were prepared with PV1 RNA and PV1VPgY3F RNA. VPg uridylylation was measured in PIRCs resuspended in reaction mixtures containing [α- 32 P]UTP. The labeled VPgpUpU synthesized in these reactions was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods and resolved by SDS-PAGE. [ 35 S]methionine-labeled poliovirus proteins were used as markers (lane 7).

    Article Snippet: The uridylylation reactions were identical to those described above for the RNA replication assays, except the reaction mixtures contained 5 μM UTP, which was provided by the addition of 100 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham) and 250 μM (each) ATP, CTP, and GTP.

    Techniques: Labeling, Synthesized, Immunoprecipitation, SDS Page

    Effect of cre (2C)mut1 on VPg uridylylation in PIRCs. Viral protein synthesis was measured in HeLa S10 translation-replication reaction mixtures containing the indicated RNAs and [ 35 S]methionine (1.2 mCi/ml) (lanes 1 to 4). VPg uridylylation was measured in PIRCs isolated from identical HeLa S10 translation-replication reaction mixtures containing each of the indicated RNAs. Reaction mixtures containing the PIRCs and [α- 32 P]UTP were incubated at 37°C for 1 h. Labeled VPgpUpU was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods (lanes 5 to 12). The labeled viral proteins and immunoprecipitated products were resolved by SDS-PAGE (9 to 18% polyacrylamide).

    Journal: Journal of Virology

    Article Title: Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

    doi: 10.1128/JVI.77.9.5136-5144.2003

    Figure Lengend Snippet: Effect of cre (2C)mut1 on VPg uridylylation in PIRCs. Viral protein synthesis was measured in HeLa S10 translation-replication reaction mixtures containing the indicated RNAs and [ 35 S]methionine (1.2 mCi/ml) (lanes 1 to 4). VPg uridylylation was measured in PIRCs isolated from identical HeLa S10 translation-replication reaction mixtures containing each of the indicated RNAs. Reaction mixtures containing the PIRCs and [α- 32 P]UTP were incubated at 37°C for 1 h. Labeled VPgpUpU was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods (lanes 5 to 12). The labeled viral proteins and immunoprecipitated products were resolved by SDS-PAGE (9 to 18% polyacrylamide).

    Article Snippet: The uridylylation reactions were identical to those described above for the RNA replication assays, except the reaction mixtures contained 5 μM UTP, which was provided by the addition of 100 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham) and 250 μM (each) ATP, CTP, and GTP.

    Techniques: Isolation, Incubation, Labeling, Immunoprecipitation, SDS Page

    Edar expression and NF-κB reporter expression co-localize in the mammary epithelium. (A) Edar transcripts were detected by in situ hybridization with a 35 S-UTP-labeled probe in mammary buds at E12.5 and E13.5. (B) NF-κB reporter was initially expressed throughout the mammary bud in control embryos, but became localized to the basal layer of the epithelium around E13.5. (C) In K14-Eda embryos, reporter activity stayed high throughout the mammary bud at E12.5 and E13.5. mb4 = mammary bud number 4. (D) The supernumerary buds exhibited mosaic expression of the reporter which was less pronounced in the in the neck (left) than in mammary primordia forming between buds 3 and 4 (right). (Scale bar: 100 μm.)

    Journal: PLoS Genetics

    Article Title: Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway

    doi: 10.1371/journal.pgen.1005676

    Figure Lengend Snippet: Edar expression and NF-κB reporter expression co-localize in the mammary epithelium. (A) Edar transcripts were detected by in situ hybridization with a 35 S-UTP-labeled probe in mammary buds at E12.5 and E13.5. (B) NF-κB reporter was initially expressed throughout the mammary bud in control embryos, but became localized to the basal layer of the epithelium around E13.5. (C) In K14-Eda embryos, reporter activity stayed high throughout the mammary bud at E12.5 and E13.5. mb4 = mammary bud number 4. (D) The supernumerary buds exhibited mosaic expression of the reporter which was less pronounced in the in the neck (left) than in mammary primordia forming between buds 3 and 4 (right). (Scale bar: 100 μm.)

    Article Snippet: Radioactive in situ hybridization on paraffin sections was carried out according to previously described protocols using 35 S-UTP labelled (Amersham) probe specific to Edar [ ].

    Techniques: Expressing, In Situ Hybridization, Labeling, Activity Assay

    Mutagenesis of rev 3′ splice site A4e of HS1-A70 does not increase splicing at the env/nef 3′ splice site A5. (A) Denaturing PAGE (6% gel) of [ 32 P]UTP-labeled HS1-A70 and rev A4e splice site mutant (HS1-A4e) substrates spliced in vitro. Also shown is in vitro splicing of NL4-3 substrate HS1-X. HS1-A4e is an AG-to-AC mutant at the A4e 3′ splice site. The positions of precursors and products spliced at the indicated splice sites are shown. (B) Quantitation of spliced tat , rev , and env/nef spliced products from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of products spliced at the tat , rev , and env/nef 3′ splice sites were calculated based on of uridine content of the different RNA species.

    Journal: Journal of Virology

    Article Title: Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

    doi:

    Figure Lengend Snippet: Mutagenesis of rev 3′ splice site A4e of HS1-A70 does not increase splicing at the env/nef 3′ splice site A5. (A) Denaturing PAGE (6% gel) of [ 32 P]UTP-labeled HS1-A70 and rev A4e splice site mutant (HS1-A4e) substrates spliced in vitro. Also shown is in vitro splicing of NL4-3 substrate HS1-X. HS1-A4e is an AG-to-AC mutant at the A4e 3′ splice site. The positions of precursors and products spliced at the indicated splice sites are shown. (B) Quantitation of spliced tat , rev , and env/nef spliced products from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of products spliced at the tat , rev , and env/nef 3′ splice sites were calculated based on of uridine content of the different RNA species.

    Article Snippet: In vitro runoff RNA transcripts labeled with [32 P]UTP (Amersham Pharmacia Biotech) were carried out as previously described ( ).

    Techniques: Mutagenesis, Polyacrylamide Gel Electrophoresis, Labeling, In Vitro, Quantitation Assay

    Comparison of tat 3′ splice site usage in different HIV-1 strains by in vitro splicing assays. (A) Denaturing PAGE of [ 32 P]UTP-labeled HIV-1 substrates spliced in vitro. The positions of precursors, spliced products, and tat lariat products are indicated. On the left is a 6% gel comparing HS1-X, HS1-SF2, and HS1-A70; on the right is a 4% gel comparing HS1-ESS4 and HS1-A70. HS1-A70 spliced product migrates more slowly because the restriction site used to produce the linearized DNA template for transcription of RNA substrates is 7 nt further downstream than for HS1-X and HS1-SF2, resulting in longer RNA products. tat lariats migrate more slowly than the linear RNA species on 6% compared to 4% gels. (B) Quantitation of spliced tat RNA from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of product spliced at the tat 3′ splice site (A3) were calculated based on uridine content of the RNA species.

    Journal: Journal of Virology

    Article Title: Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

    doi:

    Figure Lengend Snippet: Comparison of tat 3′ splice site usage in different HIV-1 strains by in vitro splicing assays. (A) Denaturing PAGE of [ 32 P]UTP-labeled HIV-1 substrates spliced in vitro. The positions of precursors, spliced products, and tat lariat products are indicated. On the left is a 6% gel comparing HS1-X, HS1-SF2, and HS1-A70; on the right is a 4% gel comparing HS1-ESS4 and HS1-A70. HS1-A70 spliced product migrates more slowly because the restriction site used to produce the linearized DNA template for transcription of RNA substrates is 7 nt further downstream than for HS1-X and HS1-SF2, resulting in longer RNA products. tat lariats migrate more slowly than the linear RNA species on 6% compared to 4% gels. (B) Quantitation of spliced tat RNA from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of product spliced at the tat 3′ splice site (A3) were calculated based on uridine content of the RNA species.

    Article Snippet: In vitro runoff RNA transcripts labeled with [32 P]UTP (Amersham Pharmacia Biotech) were carried out as previously described ( ).

    Techniques: In Vitro, Polyacrylamide Gel Electrophoresis, Labeling, Quantitation Assay

    Mutagenesis of the rev A4b 3′ AG dinucleotide enhances splicing of the env/nef 3′ splice site in HS1-SF2. (A) Denaturing PAGE (6% gel) of [ 32 P]UTP-labeled HS1-SF2 and rev A4b splice site mutant (HS1-SF4b) substrates spliced in vitro. Also shown is in vitro splicing of NL4-3 substrate HS1-X. HS1-SF4b is an AG-to-AC mutant at the A4b 3′ splice site. The positions of precursors and products spliced at the indicated splice sites are shown. (B) Quantitation of spliced tat , rev , and env/nef spliced products from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of products spliced at the tat , rev , and env/nef 3′ splice sites were calculated based on of uridine content of the different RNA species.

    Journal: Journal of Virology

    Article Title: Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

    doi:

    Figure Lengend Snippet: Mutagenesis of the rev A4b 3′ AG dinucleotide enhances splicing of the env/nef 3′ splice site in HS1-SF2. (A) Denaturing PAGE (6% gel) of [ 32 P]UTP-labeled HS1-SF2 and rev A4b splice site mutant (HS1-SF4b) substrates spliced in vitro. Also shown is in vitro splicing of NL4-3 substrate HS1-X. HS1-SF4b is an AG-to-AC mutant at the A4b 3′ splice site. The positions of precursors and products spliced at the indicated splice sites are shown. (B) Quantitation of spliced tat , rev , and env/nef spliced products from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of products spliced at the tat , rev , and env/nef 3′ splice sites were calculated based on of uridine content of the different RNA species.

    Article Snippet: In vitro runoff RNA transcripts labeled with [32 P]UTP (Amersham Pharmacia Biotech) were carried out as previously described ( ).

    Techniques: Mutagenesis, Polyacrylamide Gel Electrophoresis, Labeling, In Vitro, Quantitation Assay

    In situ localization of ILK mRNA in the skin of the transgenic mouse overexpressing the activated erbB-2. A to D: In situ localization of ILK and erbB-2 mRNAs. Frozen sections of the back skin of a 1-day-old K14-erbB-2 transgenic founder mouse (TG4839) were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled cRNA probes for the ILK ( A and B ) or the erbB-2 ( C and D ). After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. E and F: PCNA immunostaining in neonatal skins of normal ( E ) and the K14-erbB-2 ( F ) mice. Paraffin sections were subjected to PCNA immunostaining with 3,3′-diaminobenzidine tetrahydrochloride as the chromogen. The sections were counterstained with hematoxylin. Original magnification ( A to F ), ×200.

    Journal: The American Journal of Pathology

    Article Title: Expression of the Integrin-Linked Kinase (ILK) in Mouse Skin

    doi:

    Figure Lengend Snippet: In situ localization of ILK mRNA in the skin of the transgenic mouse overexpressing the activated erbB-2. A to D: In situ localization of ILK and erbB-2 mRNAs. Frozen sections of the back skin of a 1-day-old K14-erbB-2 transgenic founder mouse (TG4839) were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled cRNA probes for the ILK ( A and B ) or the erbB-2 ( C and D ). After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. E and F: PCNA immunostaining in neonatal skins of normal ( E ) and the K14-erbB-2 ( F ) mice. Paraffin sections were subjected to PCNA immunostaining with 3,3′-diaminobenzidine tetrahydrochloride as the chromogen. The sections were counterstained with hematoxylin. Original magnification ( A to F ), ×200.

    Article Snippet: High-specific activity riboprobes were synthesized in 10-μl reactions containing 100 μCi [35 S]UTP and 100 μCi [35 S]CTP (Amersham, Arlington Heights, IL), 10 mmol/L NaCl, 6 mmol/L MgCl2 , 40 mmol/L Tris (ph 7.5), 2 mmol/L spermidine, 10 mmol/L dithiothreitol, 500 μmol/L each of unlabeled ATP and GTP, 25 μmol/L each of unlabeled UTP and CTP, 0.5–1 μg linearized template, 15 U of the appropriate polymerase, and 15 U RNase inhibitor (RNasin, Promega, Madison, WI).

    Techniques: In Situ, Transgenic Assay, In Situ Hybridization, Labeling, Hybridization, Microscopy, Immunostaining, Mouse Assay

    In situ localization of ILK and PINCH mRNAs in normal mouse skins. Frozen sections of the back skin of 1-day-old mouse were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled ILK ( A and B ) and PINCH ( C and D ) cRNA probes, respectively. After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. SC, stratum corneum; GR, granular cells; SP, spinous cells; BL, basal layer; hf, hair follicle. Arrows indicate hair follicles. Original magnification ( A to D ), ×200.

    Journal: The American Journal of Pathology

    Article Title: Expression of the Integrin-Linked Kinase (ILK) in Mouse Skin

    doi:

    Figure Lengend Snippet: In situ localization of ILK and PINCH mRNAs in normal mouse skins. Frozen sections of the back skin of 1-day-old mouse were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled ILK ( A and B ) and PINCH ( C and D ) cRNA probes, respectively. After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. SC, stratum corneum; GR, granular cells; SP, spinous cells; BL, basal layer; hf, hair follicle. Arrows indicate hair follicles. Original magnification ( A to D ), ×200.

    Article Snippet: High-specific activity riboprobes were synthesized in 10-μl reactions containing 100 μCi [35 S]UTP and 100 μCi [35 S]CTP (Amersham, Arlington Heights, IL), 10 mmol/L NaCl, 6 mmol/L MgCl2 , 40 mmol/L Tris (ph 7.5), 2 mmol/L spermidine, 10 mmol/L dithiothreitol, 500 μmol/L each of unlabeled ATP and GTP, 25 μmol/L each of unlabeled UTP and CTP, 0.5–1 μg linearized template, 15 U of the appropriate polymerase, and 15 U RNase inhibitor (RNasin, Promega, Madison, WI).

    Techniques: In Situ, In Situ Hybridization, Labeling, Hybridization, Microscopy

    Demonstration of CENP-E messenger RNA and protein in rheumatoid synovium by in situ hybridization and immunohistochemistry. (a, b) A digoxigenin-labelled RNA probe transcribed from the amplified RNA arbitrarily primed-PCR gene product is used on rheumatoid arthritis snap-frozen sections. Double-labelling for CENP-E messenger RNA (black staining), and alkaline phosphatase antialkaline phosphatase counterstaining using anti-fibroblast antibodies (red staining) shows CENP-E expression in numerous fibroblasts througout the synovium (a, arrows). (b) The intensive CENP-E messenger RNA expression in two synovial fibroblasts. (c) Shows numerous fibroblasts expressing CENP-E protein [black staining (arrows), counterstaining with fast red]. Original magnifications × 300 (a, c) and × 600 (b).

    Journal: Arthritis Research

    Article Title: Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts

    doi:

    Figure Lengend Snippet: Demonstration of CENP-E messenger RNA and protein in rheumatoid synovium by in situ hybridization and immunohistochemistry. (a, b) A digoxigenin-labelled RNA probe transcribed from the amplified RNA arbitrarily primed-PCR gene product is used on rheumatoid arthritis snap-frozen sections. Double-labelling for CENP-E messenger RNA (black staining), and alkaline phosphatase antialkaline phosphatase counterstaining using anti-fibroblast antibodies (red staining) shows CENP-E expression in numerous fibroblasts througout the synovium (a, arrows). (b) The intensive CENP-E messenger RNA expression in two synovial fibroblasts. (c) Shows numerous fibroblasts expressing CENP-E protein [black staining (arrows), counterstaining with fast red]. Original magnifications × 300 (a, c) and × 600 (b).

    Article Snippet: Probes were labelled with digoxigenin-uridine triphosphate (Boehringer Mannheim).

    Techniques: In Situ Hybridization, Immunohistochemistry, Amplification, Polymerase Chain Reaction, Staining, Expressing, RNA Expression

    In vitro RdRp assay with exogenous RNA templates. Approximately 50 ng of RNA template r138/40A (the 3′ UTR of Bamboo mosaic virus (BaMV)) in (a) and Ba-77 (the 3′-end 77 nts of BaMV minus-strand) in (b) were incubated with BaMV RdRp complex for the in vitro RNA synthesis in the presence of different concentrations (0–100 mM) of glutathione (GSH) as indicated. The RdRp products labeled with [α- 32 P] UTP as indicated bands were separated on a 5% acrylamide gel and quantified by a phosphorimager. (c) The relative RdRp template activities were plotted according to the data derived from (a) and (b). The banding density of the in vitro RdRp assay with either r138/40A or Ba-77 was set as 100% in the absence of GSH (0 mM). Each spot on the plot was the average ± SE of at least three independent experiments.

    Journal: The New Phytologist

    Article Title: The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus

    doi: 10.1111/nph.12304

    Figure Lengend Snippet: In vitro RdRp assay with exogenous RNA templates. Approximately 50 ng of RNA template r138/40A (the 3′ UTR of Bamboo mosaic virus (BaMV)) in (a) and Ba-77 (the 3′-end 77 nts of BaMV minus-strand) in (b) were incubated with BaMV RdRp complex for the in vitro RNA synthesis in the presence of different concentrations (0–100 mM) of glutathione (GSH) as indicated. The RdRp products labeled with [α- 32 P] UTP as indicated bands were separated on a 5% acrylamide gel and quantified by a phosphorimager. (c) The relative RdRp template activities were plotted according to the data derived from (a) and (b). The banding density of the in vitro RdRp assay with either r138/40A or Ba-77 was set as 100% in the absence of GSH (0 mM). Each spot on the plot was the average ± SE of at least three independent experiments.

    Article Snippet: The RdRp assay was carried out in a 50-μl reaction containing 5 μl of 1.5% NP40-solublized RdRp fraction, 2 mM each of ATP, CTP, and GTP, 2 μM UTP, 0.066 μM [α-32 P]UTP (3000 Ci mmol−1 , Dupont-NEN), 4.8 mg ml−1 of bentonite, 10 mM dithiothreitol, 3 mM MgCl2 , and 50 ng of RNA template and incubated at 30°C water bath for 1 h. The RNA products were extracted with phenol-chloroform and precipitated with ethanol.

    Techniques: In Vitro, Incubation, Labeling, Acrylamide Gel Assay, Derivative Assay

    Expression pattern of the zen ( A and B ) and twist ( C and D ) genes in blastoderm embryos. Expression is visualised by whole mount in situ hybridisation using a digoxigenin-11-UTP-labelled antisense RNA probe followed by immunological staining. Embryos are oriented with anterior to the left and dorsal up (A and B) or in ventral view (C and D). (A) and (C), wild-type embryos; (B) and (D), DSP1 mutant embryos.

    Journal: Nucleic Acids Research

    Article Title: HMG boxes of DSP1 protein interact with the Rel homology domain of transcription factors

    doi:

    Figure Lengend Snippet: Expression pattern of the zen ( A and B ) and twist ( C and D ) genes in blastoderm embryos. Expression is visualised by whole mount in situ hybridisation using a digoxigenin-11-UTP-labelled antisense RNA probe followed by immunological staining. Embryos are oriented with anterior to the left and dorsal up (A and B) or in ventral view (C and D). (A) and (C), wild-type embryos; (B) and (D), DSP1 mutant embryos.

    Article Snippet: The probes used were a zen or twist antisense riboprobe labelled with digoxigenin-11-UTP (Boehringer Mannheim Biochemicals).

    Techniques: Expressing, In Situ, Hybridization, Staining, Mutagenesis

    Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate (UTP) in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.

    Journal: British Journal of Pharmacology

    Article Title: Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia

    doi: 10.1111/j.1476-5381.2010.00736.x

    Figure Lengend Snippet: Effects of extracellular divalent cations (DICs) on P2X receptor agonist potencies as well as effects of various P2X and P2Y receptor agonists in rat cultured cortical astroglia. (A) Concentration-response curves for the peak currents induced by ATP (3–10 000 µM) and 2′-3′-O-(4-benzoyl) ATP (BzATP) (0.3–1000 µM) in rat cortical astrocytes recorded either with standard concentrations of extracellular DICs (standard DIC; 1.2 mM Mg 2+ and 1.0 mM Ca 2+ ) or in a low DIC-containing bath (low DIC; 0 mM added Mg 2+ , 0.1 mM Ca 2+ ). The continuous lines were obtained by fitting a three-parameter logistic function (Hill equation) to the averaged data ( n = 9–13, N = 3–4). Concentration-response curves were constructed from currents in response to subsequently increasing drug concentrations, pressure applied for 3 s at a holding potential of −80 mV, which were separated by a 4 min washout with the respective agonist-free bath solutions (standard bath or low DIC). The insets show, from left to right, representative superimposed whole-cell currents evoked by increasing concentrations of ATP in standard DIC, ATP in low DIC, BzATP in standard DIC and BzATP in low DIC. (B) Representative whole-cell currents evoked by 300 µM of BzATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2-(methylthio) ATP (2MeSATP), drug-free bath solution (saline), uridine 5′-diphosphate (UDP), α,β-methylene ATP (α,βmeATP), ATP, uridine 5′-diphosphoglucose (UDP-Gluc) and uridine 5′-triphosphate (UTP) in a rat cortical astroglial cell. P2 agonists were consecutively applied (for 3 s every 4 min at a holding potential of −80 mV) to the same set of cells. The order of agonist application was varied between individual trials. As in (A), a Cs + -based pipette solution was used. The bath solution was low DIC to allow for the detection of even small events.

    Article Snippet: The following chemicals were used: AMPA, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), muscimol and tetrodotoxin (TTX) (Biotrend, Köln, Germany); Triton X-100 (Carl Roth GmbH); Dulbecco's modified Eagle's medium plus nutrient mixture F12 (1:1) plus 2.5 mM L-alanyl-L-glutamine (Dulbecco's modified Eagle's medium/F12 1:1 with GlutaMAX™ I), HBSS, Hoe, rabbit anti-LY antibody and trypsin (Invitrogen); Cy5-conjugated donkey anti-mouse IgG and Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch); FCS (Seromed); 2-(methylthio) ATP (2MeSATP), adenosine 5′-O-(2-thiodiphosphate) (ADPβS), ATP, α,β-methylene ATP (α,βmeATP), ATP periodate oxidized (oATP), BzATP, calmidazolium (CAL), CBX, Coomassie Brilliant Blue G (BBG), cytosine-β- d -arabinofuranosid, gentamicin, ivermectin (IVM), LY, mouse anti-GFAP antibody, poly-L-lysine, pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), suramin, 2′-3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), uridine 5′-diphosphate (UDP), uridine 5′-diphosphoglucose (UDP-Gluc), uridine 5′-triphosphate (UTP) and ZnCl (Sigma-Aldrich).

    Techniques: Cell Culture, Concentration Assay, Construct, Transferring

    aRNA yield after different incubation times. 5 μg of total RNA were amplified and labeled (either Cy3-UTP or Cy5-UTP) or non-labeled ('no Dye' control) with the T7-System for 2, 4, 6, 8 and 16 hours (n = 3). The aRNA was purified by column chromatography and quantified by UV-spectrophotometry.

    Journal: BMC Genomics

    Article Title: Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

    doi: 10.1186/1471-2164-4-44

    Figure Lengend Snippet: aRNA yield after different incubation times. 5 μg of total RNA were amplified and labeled (either Cy3-UTP or Cy5-UTP) or non-labeled ('no Dye' control) with the T7-System for 2, 4, 6, 8 and 16 hours (n = 3). The aRNA was purified by column chromatography and quantified by UV-spectrophotometry.

    Article Snippet: The cDNA was concentrated to 3 μl in a vacuum centrifuge and the in-vitro transcription (IVT) was carried out for the indicated incubation times (2 h, 4 h, 6 h, 8 h and 16 h) at 37°C in a reaction mixture (20 μl) containing 2 μl 10× amplification buffer, 2 μl each ATP, CTP, GTP, UTP (75 mM each) and 5 μl Cy3-UTP or Cy5-UTP (5 mM, Amersham Pharmacia Biotech, Sunnyvale, CA).

    Techniques: Incubation, Amplification, Labeling, Purification, Column Chromatography, Spectrophotometry

    Incubation-time dependent degradation of aRNA. 5 μg of total RNA were amplified and labeled (either with Cy3-UTP or Cy5-UTP) with the T7-System for 2, 4, 6, 8 and 16 hours. The aRNA was separated on a denaturing agarose gel and photographed on a gel-imaging system. Fainter bands in the Cy5-labeled aRNA are due to quenching of Cy5-fluorescence by SybrGreen II and lower yield. M=Molecular weight marker.

    Journal: BMC Genomics

    Article Title: Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

    doi: 10.1186/1471-2164-4-44

    Figure Lengend Snippet: Incubation-time dependent degradation of aRNA. 5 μg of total RNA were amplified and labeled (either with Cy3-UTP or Cy5-UTP) with the T7-System for 2, 4, 6, 8 and 16 hours. The aRNA was separated on a denaturing agarose gel and photographed on a gel-imaging system. Fainter bands in the Cy5-labeled aRNA are due to quenching of Cy5-fluorescence by SybrGreen II and lower yield. M=Molecular weight marker.

    Article Snippet: The cDNA was concentrated to 3 μl in a vacuum centrifuge and the in-vitro transcription (IVT) was carried out for the indicated incubation times (2 h, 4 h, 6 h, 8 h and 16 h) at 37°C in a reaction mixture (20 μl) containing 2 μl 10× amplification buffer, 2 μl each ATP, CTP, GTP, UTP (75 mM each) and 5 μl Cy3-UTP or Cy5-UTP (5 mM, Amersham Pharmacia Biotech, Sunnyvale, CA).

    Techniques: Incubation, Amplification, Labeling, Agarose Gel Electrophoresis, Imaging, Fluorescence, Molecular Weight, Marker

    Affinity-purified ytmQ gene product (BsTrmB) catalyzes the formation of m 7 G in T7 transcripts of B.subtilis tRNA Phe . ( A ) B.subtilis tRNA Phe (5 µg) was incubated with 5 µg of purified BsTrmB protein and 15 µM of [methyl- 14 C]AdoMet (58 mCi/mmol) in TM buffer (50 mM Tris–HCl and 10 mM MgCl 2 , pH8) in a total volume of 200 µl. After 30 min incubation at 37°C the tRNA was recovered and digested with nuclease P1. The resulting nucleotides were analyzed by 2D-TLC and autoradiography as described ( 42 ). First dimension developed with isobutyric acid/conc. NH 4 OH/water (66:1:33; v/v/v); second dimension developed with conc. HCl/isopropanol/water (17.6:68:14.4; v/v/v). ( B ) [α- 32 P]UTP-labeled in vitro transcribed B.subtilis tRNA Phe (5 × 10 5 c.p.m.) was incubated with 5 µg of purified BsTrmB and 30 µM AdoMet in TM buffer in a total volume of 300 µl. After 30 min incubation at 37°C, the tRNA was recovered, hydrolyzed by RNAse T2 and the resulting nucleotides were analyzed as above.

    Journal: Nucleic Acids Research

    Article Title: Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase

    doi: 10.1093/nar/gkl116

    Figure Lengend Snippet: Affinity-purified ytmQ gene product (BsTrmB) catalyzes the formation of m 7 G in T7 transcripts of B.subtilis tRNA Phe . ( A ) B.subtilis tRNA Phe (5 µg) was incubated with 5 µg of purified BsTrmB protein and 15 µM of [methyl- 14 C]AdoMet (58 mCi/mmol) in TM buffer (50 mM Tris–HCl and 10 mM MgCl 2 , pH8) in a total volume of 200 µl. After 30 min incubation at 37°C the tRNA was recovered and digested with nuclease P1. The resulting nucleotides were analyzed by 2D-TLC and autoradiography as described ( 42 ). First dimension developed with isobutyric acid/conc. NH 4 OH/water (66:1:33; v/v/v); second dimension developed with conc. HCl/isopropanol/water (17.6:68:14.4; v/v/v). ( B ) [α- 32 P]UTP-labeled in vitro transcribed B.subtilis tRNA Phe (5 × 10 5 c.p.m.) was incubated with 5 µg of purified BsTrmB and 30 µM AdoMet in TM buffer in a total volume of 300 µl. After 30 min incubation at 37°C, the tRNA was recovered, hydrolyzed by RNAse T2 and the resulting nucleotides were analyzed as above.

    Article Snippet: [α-32 P]GTP and [α-32 P]UTP were purchased from MPBiomedicals and T7 RNA polymerase from Roche Applied Science.

    Techniques: Affinity Purification, Incubation, Purification, Thin Layer Chromatography, Autoradiography, Labeling, In Vitro

    Co-localization of endogenous Staufen protein and injected VLE RNA. Stage II oocytes were injected with 500 nM Cy5-UTP-labeled VLE ) at 1:250 dilution and an Alexa-546-secondary antibody (Thermo-Fisher) at 1:500 dilution. Oocytes were imaged on a Zeiss LSM 510 Meta Confocal Laser Scanning Microscope using a 20× objective. Shown is a confocal section with (A) VLE RNA in red, (B) Staufen protein in green, and (C) merged red and green channels, showing Staufen protein and VLE RNA colocalization. The oocyte is oriented with the vegetal pole at the bottom and the scale bars = 50 µm.

    Journal: Cold Spring Harbor protocols

    Article Title: WHOLE-MOUNT IMMUNOFLUORESCENCE FOR VISUALIZING ENDOGENOUS PROTEIN AND INJECTED RNA IN XENOPUS OOCYTES

    doi: 10.1101/pdb.prot097022

    Figure Lengend Snippet: Co-localization of endogenous Staufen protein and injected VLE RNA. Stage II oocytes were injected with 500 nM Cy5-UTP-labeled VLE ) at 1:250 dilution and an Alexa-546-secondary antibody (Thermo-Fisher) at 1:500 dilution. Oocytes were imaged on a Zeiss LSM 510 Meta Confocal Laser Scanning Microscope using a 20× objective. Shown is a confocal section with (A) VLE RNA in red, (B) Staufen protein in green, and (C) merged red and green channels, showing Staufen protein and VLE RNA colocalization. The oocyte is oriented with the vegetal pole at the bottom and the scale bars = 50 µm.

    Article Snippet: Ammonium acetate (7 M, RNase-free) Collagenase solution < R > Cy5-UTP or Cy3-UTP (GE Healthcare Life Sciences, PA55026/PA53026) Ethanol (70%, RNase-free) Ethanol (100%, RNase-free) Glycogen (20 mg/mL; Thermo Scientific, R0561) MBSH Buffer (10×) < R > MEMFA < R > Methanol (anhydrous, 100%) mMessage machine SP6/T7/T3 transcription kit (Ambion, AM1340/AM1344/AM1348) Murray’s clear < R > Nuclease-free H2 O (Thermo Fisher, AM9914G) or H2 O treated with DEPC (Sigma D5758) Oocyte culture medium (OCM) < R > PBT for Xenopus oocytes/PBT-Plus < R > Phenol/chloroform/isoamylalcohol (25:24:1, Fisher Scientific, AC327115000) Plasmid containing sequence of interest downstream of an RNA polymerase promoter (T7, SP6, or T3) Proteinase K buffer for Xenopus oocytes < R > Primary antibody Restriction enzyme appropriate for plasmid linearization RNase-ZAP (Ambion, AM9780) Secondary antibody, fluorescently-labeled

    Techniques: Injection, Labeling, Laser-Scanning Microscopy