dctp  (Thermo Fisher)


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    Structured Review

    Thermo Fisher dctp
    The allosteric sites of SAMHD1 are highly restrictive. ( A ) Size-exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM <t>GTP</t> and 4 mM of color-coded nucleotide analog. ( B ) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 μM. ( C ) Malachite green activity assay performed in the presence of 125 μM GTP and 125 μM <t>dCTP,</t> dATP, nucleotide analog, or buffer. Error bars represent SEM of three independent experiments. ( D ) Malachite green activity assay measuring the hydrolysis of dATP by SAMHD1 tetramers preassembled in the presence of 125 μM GTP and 6.3 to 3,200 μM dATP, cladribine-TP, clofarabine-TP, or ATP. Error bars represent SEM of three independent experiments.
    Dctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dctp - by Bioz Stars, 2022-08
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    Images

    1) Product Images from "The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1"

    Article Title: The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1805593115

    The allosteric sites of SAMHD1 are highly restrictive. ( A ) Size-exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM GTP and 4 mM of color-coded nucleotide analog. ( B ) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 μM. ( C ) Malachite green activity assay performed in the presence of 125 μM GTP and 125 μM dCTP, dATP, nucleotide analog, or buffer. Error bars represent SEM of three independent experiments. ( D ) Malachite green activity assay measuring the hydrolysis of dATP by SAMHD1 tetramers preassembled in the presence of 125 μM GTP and 6.3 to 3,200 μM dATP, cladribine-TP, clofarabine-TP, or ATP. Error bars represent SEM of three independent experiments.
    Figure Legend Snippet: The allosteric sites of SAMHD1 are highly restrictive. ( A ) Size-exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM GTP and 4 mM of color-coded nucleotide analog. ( B ) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 μM. ( C ) Malachite green activity assay performed in the presence of 125 μM GTP and 125 μM dCTP, dATP, nucleotide analog, or buffer. Error bars represent SEM of three independent experiments. ( D ) Malachite green activity assay measuring the hydrolysis of dATP by SAMHD1 tetramers preassembled in the presence of 125 μM GTP and 6.3 to 3,200 μM dATP, cladribine-TP, clofarabine-TP, or ATP. Error bars represent SEM of three independent experiments.

    Techniques Used: Size-exclusion Chromatography, Concentration Assay, Activity Assay

    2) Product Images from "Substrates and Inhibitors of SAMHD1"

    Article Title: Substrates and Inhibitors of SAMHD1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169052

    Role of Y374 and C2' sugar moiety substitution in acting as substrates of SAMHD1. A) dCTP, C) ara-CTP and E) SMDU-TP nucleotides (in green) are modeled within the catalytic site of SAMHD1. Both dCTP and ara-CTP do not clash with Y374 (see arrow). However the model shows that the (2' S )-2'-methyl group of SMDU-TP clashes with Y374 in the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, ara-CTP and SMDU-TP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs with experimental conditions described in Fig 2 . Data are presented as the percent compound remaining (y-axis). dCTP and ara-CTP are significantly hydrolyzed (p
    Figure Legend Snippet: Role of Y374 and C2' sugar moiety substitution in acting as substrates of SAMHD1. A) dCTP, C) ara-CTP and E) SMDU-TP nucleotides (in green) are modeled within the catalytic site of SAMHD1. Both dCTP and ara-CTP do not clash with Y374 (see arrow). However the model shows that the (2' S )-2'-methyl group of SMDU-TP clashes with Y374 in the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, ara-CTP and SMDU-TP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs with experimental conditions described in Fig 2 . Data are presented as the percent compound remaining (y-axis). dCTP and ara-CTP are significantly hydrolyzed (p

    Techniques Used: Acetylene Reduction Assay, In Vitro

    Monitoring ara-CTP and gem-TP concentrations in MDMs. A) Monocyte-derived macrophages (MDMs) were pretreated with virus-like particles (VLP) one day prior to replacing the medium with fresh medium plus compounds: 10 μM of ara-C (cytaribine- 13 C 3 ) or 10 μM of gemcitabine. Whole cell lysates were collected at 0, 24 and 48 h post VLP addition. Lysates were analyzed by immunoblotting for SAMHD1 and GAPDH, loading control. SAMHD1 protein levels were reduced at 24 h after Vpx+ VLP exposure. Two human primary MDM donors are shown. Cellular nucleotide extracts were generated at 4, 12 and 24 h post drug addition from treated MDMs. The intracellular concentrations of B) gem-TP (2',2'-diF-dCTP) and (C) ara-CTP were quantified from the extracts using HPLC-MS/MS analysis. Data are plotted as pmol/million cells (y-axis) vs . time (h) (x-axis). Gem-TP is a significantly lower (**, p
    Figure Legend Snippet: Monitoring ara-CTP and gem-TP concentrations in MDMs. A) Monocyte-derived macrophages (MDMs) were pretreated with virus-like particles (VLP) one day prior to replacing the medium with fresh medium plus compounds: 10 μM of ara-C (cytaribine- 13 C 3 ) or 10 μM of gemcitabine. Whole cell lysates were collected at 0, 24 and 48 h post VLP addition. Lysates were analyzed by immunoblotting for SAMHD1 and GAPDH, loading control. SAMHD1 protein levels were reduced at 24 h after Vpx+ VLP exposure. Two human primary MDM donors are shown. Cellular nucleotide extracts were generated at 4, 12 and 24 h post drug addition from treated MDMs. The intracellular concentrations of B) gem-TP (2',2'-diF-dCTP) and (C) ara-CTP were quantified from the extracts using HPLC-MS/MS analysis. Data are plotted as pmol/million cells (y-axis) vs . time (h) (x-axis). Gem-TP is a significantly lower (**, p

    Techniques Used: Acetylene Reduction Assay, Derivative Assay, Generated, High Performance Liquid Chromatography, Mass Spectrometry

    Ara-CTP does not fit into the A2 site of SAMHD1. A) Evaluating ara-CTP hydrolysis in the presence of dGTP, using as A1site activator. When dGTP was present, ara-CTP and dCTP were significantly hydrolyzed (p
    Figure Legend Snippet: Ara-CTP does not fit into the A2 site of SAMHD1. A) Evaluating ara-CTP hydrolysis in the presence of dGTP, using as A1site activator. When dGTP was present, ara-CTP and dCTP were significantly hydrolyzed (p

    Techniques Used: Acetylene Reduction Assay

    Examining the role of L150 for nucleotide specificity. A) dCTP, C) (2' R ) 2'-F-dCTP and E) CTP nucleotides (in green) are modeled in the catalytic site of SAMHD1. L150 clashes with (2' R ) 2'-F-dCTP and CTP, but not dCTP (see arrows) within the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, (2' R ) 2'-F-dCTP and CTP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs. Using semi-quantitative HLPC analysis method, compounds were incubated with and without 1.6 μM of SAMHD1 enzyme plus dGTP (A1 site activator) to determine if they are substrates of SAMHD1. Data are presented as the percent compound remaining (y-axis). dCTP and dGTP were significantly hydrolyzed (p
    Figure Legend Snippet: Examining the role of L150 for nucleotide specificity. A) dCTP, C) (2' R ) 2'-F-dCTP and E) CTP nucleotides (in green) are modeled in the catalytic site of SAMHD1. L150 clashes with (2' R ) 2'-F-dCTP and CTP, but not dCTP (see arrows) within the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, (2' R ) 2'-F-dCTP and CTP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs. Using semi-quantitative HLPC analysis method, compounds were incubated with and without 1.6 μM of SAMHD1 enzyme plus dGTP (A1 site activator) to determine if they are substrates of SAMHD1. Data are presented as the percent compound remaining (y-axis). dCTP and dGTP were significantly hydrolyzed (p

    Techniques Used: In Vitro, Incubation

    3) Product Images from "IMP/GTP balance modulates cytoophidium assembly and IMPDH activity"

    Article Title: IMP/GTP balance modulates cytoophidium assembly and IMPDH activity

    Journal: Cell Division

    doi: 10.1186/s13008-018-0038-0

    IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments
    Figure Legend Snippet: IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments

    Techniques Used:

    4) Product Images from "An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis"

    Article Title: An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

    Journal: Nucleic Acids Research

    doi:

    Scatter plots of experiments with total RNA isolated from mouse neural stem cell cultures (neurospheres, NS). The same RNA sample was used for cDNA synthesis and labeled with either fluorescein-12-dCTP or biotin-11-dCTP. Both cDNAs were co-hybridized on the mouse 9k microarray (homotypic hybridization) and developed according to the TSA protocol provided by the manufacturer (MICROMAX; NEN). ( A ) The plot (on a log 10 scale) of Cy5 signal versus background from a representative homotypic hybridization of NS RNA on the mouse 9k array. The plots for Cy3 were similar. The points above the solid line have a signal higher than 2-fold background. The letter E indicates the signal produced by negative control spots. Notable is the clear separation between signal and background apparent in the two clusters of spots that are observed when the data are plotted in this manner. ( B ) A representative plot of the log 10 ratio (Cy3/Cy5) versus the average log 10 signal from both Cy3 and Cy5 channels for each spot in homotypic hybridization. It can be seen that the likelihood of false positive ratios decreases with signal strength. ( C ) Scatter plots of the signal generated from NS versus NS hybridizations from two experiments where the dyes were reversed. In this case, rather than labeling the same RNA, each NS culture, NS7 and NS8, was grown in separate flasks and processed separately. The clear non-linearity (curvature) produced by differences in dye incorporation and signal at different cDNA abundances can be observed.
    Figure Legend Snippet: Scatter plots of experiments with total RNA isolated from mouse neural stem cell cultures (neurospheres, NS). The same RNA sample was used for cDNA synthesis and labeled with either fluorescein-12-dCTP or biotin-11-dCTP. Both cDNAs were co-hybridized on the mouse 9k microarray (homotypic hybridization) and developed according to the TSA protocol provided by the manufacturer (MICROMAX; NEN). ( A ) The plot (on a log 10 scale) of Cy5 signal versus background from a representative homotypic hybridization of NS RNA on the mouse 9k array. The plots for Cy3 were similar. The points above the solid line have a signal higher than 2-fold background. The letter E indicates the signal produced by negative control spots. Notable is the clear separation between signal and background apparent in the two clusters of spots that are observed when the data are plotted in this manner. ( B ) A representative plot of the log 10 ratio (Cy3/Cy5) versus the average log 10 signal from both Cy3 and Cy5 channels for each spot in homotypic hybridization. It can be seen that the likelihood of false positive ratios decreases with signal strength. ( C ) Scatter plots of the signal generated from NS versus NS hybridizations from two experiments where the dyes were reversed. In this case, rather than labeling the same RNA, each NS culture, NS7 and NS8, was grown in separate flasks and processed separately. The clear non-linearity (curvature) produced by differences in dye incorporation and signal at different cDNA abundances can be observed.

    Techniques Used: Isolation, Labeling, Microarray, Hybridization, Produced, Negative Control, Generated

    5) Product Images from "Benzoate Mediates Repression of C4-Dicarboxylate Utilization in " Aromatoleum aromaticum" EbN1"

    Article Title: Benzoate Mediates Repression of C4-Dicarboxylate Utilization in " Aromatoleum aromaticum" EbN1

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.05072-11

    Variation of dctP ( ebA4158 ) transcript abundance during anaerobic diauxic growth of “ A. aromaticum ” EbN1 with succinate plus benzoate (phases 1 to 3), compared to that in cells grown anaerobically with benzoate or succinate (½
    Figure Legend Snippet: Variation of dctP ( ebA4158 ) transcript abundance during anaerobic diauxic growth of “ A. aromaticum ” EbN1 with succinate plus benzoate (phases 1 to 3), compared to that in cells grown anaerobically with benzoate or succinate (½

    Techniques Used:

    6) Product Images from "A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1"

    Article Title: A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020208

    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
    Figure Legend Snippet: Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value

    Techniques Used: DNA Synthesis

    Multiple incorrect nt incorporations at the K65 position with subtype C RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product is observed as a single dGTP, dCTP or dTTP incorporation occurred opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.
    Figure Legend Snippet: Multiple incorrect nt incorporations at the K65 position with subtype C RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product is observed as a single dGTP, dCTP or dTTP incorporation occurred opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.

    Techniques Used: DNA Synthesis, Mutagenesis

    7) Product Images from "Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes"

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks316

    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.
    Figure Legend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Techniques Used: Purification, Polymerase Chain Reaction, Construct

    Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .
    Figure Legend Snippet: Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

    Techniques Used: Polymerase Chain Reaction, Lysis, Derivative Assay, Activity Assay, Real-time Polymerase Chain Reaction, Generated

    8) Product Images from "The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1"

    Article Title: The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

    Journal: bioRxiv

    doi: 10.1101/296624

    Gemcitabine-TP but not ((2’R)-2’-F)-dCTP is hydrolyzed by SAMHD1 in vitro . (A) ((2’S)-2’-OH)) of cytarabine-TP is stabilized by residues Y374 and Y315 through van der Waals interactions. Transparent surface of SAMHD1 is shown with key residues in sticks. (B) 2’,2’-difluorine sugar modification of gemcitabine-TP is stabilized by Van der Waals interactions with residues Y374 and Y315 to compensate potential close contact (yellow caution triangle) between ((2’R)-2’-F) atom and residue L150 in the catalytic site. (C) Left: dNTPase activity of SAMHD1 over the course of 30 minutes was measured using a malachite green assay. Product is normalized to SAMHD1 concentration (nmol PO 4 / nmol SAMHD1). SAMHD1 tetramers were pre-assembled with 250 uM GTP and dATP and then diluted 100-fold into 125 uM gemcitabine-TP, dCTP, CTP, ((2’S)-2’-F)-dCTP, ((2’R)-2’-F)-dCTP, or buffer. Error bars represent standard error of the mean (SEM) of three independent experiments. Right: chemical structures of ((2’S)-2’-F)-dCTP and ((2’R)-2’-F)-dCTP analogues. (D) ((2’R)-2’-F)-dCTP (gray sticks) modeled into the catalytic pocket potentially clashes (red cross) with residue L150.
    Figure Legend Snippet: Gemcitabine-TP but not ((2’R)-2’-F)-dCTP is hydrolyzed by SAMHD1 in vitro . (A) ((2’S)-2’-OH)) of cytarabine-TP is stabilized by residues Y374 and Y315 through van der Waals interactions. Transparent surface of SAMHD1 is shown with key residues in sticks. (B) 2’,2’-difluorine sugar modification of gemcitabine-TP is stabilized by Van der Waals interactions with residues Y374 and Y315 to compensate potential close contact (yellow caution triangle) between ((2’R)-2’-F) atom and residue L150 in the catalytic site. (C) Left: dNTPase activity of SAMHD1 over the course of 30 minutes was measured using a malachite green assay. Product is normalized to SAMHD1 concentration (nmol PO 4 / nmol SAMHD1). SAMHD1 tetramers were pre-assembled with 250 uM GTP and dATP and then diluted 100-fold into 125 uM gemcitabine-TP, dCTP, CTP, ((2’S)-2’-F)-dCTP, ((2’R)-2’-F)-dCTP, or buffer. Error bars represent standard error of the mean (SEM) of three independent experiments. Right: chemical structures of ((2’S)-2’-F)-dCTP and ((2’R)-2’-F)-dCTP analogues. (D) ((2’R)-2’-F)-dCTP (gray sticks) modeled into the catalytic pocket potentially clashes (red cross) with residue L150.

    Techniques Used: In Vitro, Modification, Activity Assay, Malachite Green Assay, Concentration Assay

    The catalytic and allosteric pockets exhibit different preferences for nucleotides. (A) 2F o -F c electron density (σ = 1.0) in the catalytic pocket of SAMHD1. No electron density is observed for ((2’R)-2’-F)-dCTP (modeled as grey sticks). (B) dNTPase assay measuring triphosphates produced by pre-assembled SAMHD1 tetramer in the presence of 125 uM dCTP, dATP, nucleotide analogues, or buffer alone. Error bars represent SEM. (C) 2F o -F c electron density (σ = 1.0) for gemcitabine-TP in the catalytic pocket with labeled residues shown in sticks and black stars indicate the sites of modifications. (D) dNTPase assay measuring triphosphates produced by SAMHD1 mixed with 125 uM dGTP, dCTP, dATP, nucleotide analogues, or buffer alone. Error bars represent SEM of three independent experiments.
    Figure Legend Snippet: The catalytic and allosteric pockets exhibit different preferences for nucleotides. (A) 2F o -F c electron density (σ = 1.0) in the catalytic pocket of SAMHD1. No electron density is observed for ((2’R)-2’-F)-dCTP (modeled as grey sticks). (B) dNTPase assay measuring triphosphates produced by pre-assembled SAMHD1 tetramer in the presence of 125 uM dCTP, dATP, nucleotide analogues, or buffer alone. Error bars represent SEM. (C) 2F o -F c electron density (σ = 1.0) for gemcitabine-TP in the catalytic pocket with labeled residues shown in sticks and black stars indicate the sites of modifications. (D) dNTPase assay measuring triphosphates produced by SAMHD1 mixed with 125 uM dGTP, dCTP, dATP, nucleotide analogues, or buffer alone. Error bars represent SEM of three independent experiments.

    Techniques Used: Produced, Labeling

    The allosteric sites of SAMHD1 are highly restrictive. (A) Size exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM GTP and 4 mM of color-coded nucleotide analogue. (B) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 uM. (C) dNTPase assay performed in the presence of 125 uM GTP and 125 uM dCTP, dATP, nucleotide analogue, or buffer. Error bars represent SEM of three independent experiments.
    Figure Legend Snippet: The allosteric sites of SAMHD1 are highly restrictive. (A) Size exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM GTP and 4 mM of color-coded nucleotide analogue. (B) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 uM. (C) dNTPase assay performed in the presence of 125 uM GTP and 125 uM dCTP, dATP, nucleotide analogue, or buffer. Error bars represent SEM of three independent experiments.

    Techniques Used: Size-exclusion Chromatography, Concentration Assay

    9) Product Images from "DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications"

    Article Title: DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0260428

    Selective incorporation of labeled nucleotides. a) DNA molecules embedded in gel were incubated with a nicking enzyme. The sequence downstream of the nick is CACTG. b) A first incorporation reaction was carried out with polymerase, dCTP and labeled dUTP. Only a few dots are visible. c) Second incorporation reaction with dATP and labeled dUTP. d) The same reaction shown in Fig 4B was repeated (dCTP and dUTP). This time, many dots appeared. e) The previous reaction (dCTP and dUTP) was repeated. f) Final incorporation reaction including dGTP, dATP, dCTP and labeled dUTP.
    Figure Legend Snippet: Selective incorporation of labeled nucleotides. a) DNA molecules embedded in gel were incubated with a nicking enzyme. The sequence downstream of the nick is CACTG. b) A first incorporation reaction was carried out with polymerase, dCTP and labeled dUTP. Only a few dots are visible. c) Second incorporation reaction with dATP and labeled dUTP. d) The same reaction shown in Fig 4B was repeated (dCTP and dUTP). This time, many dots appeared. e) The previous reaction (dCTP and dUTP) was repeated. f) Final incorporation reaction including dGTP, dATP, dCTP and labeled dUTP.

    Techniques Used: Labeling, Incubation, Sequencing

    Selective incorporation of labeled nucleotides. a) DNA molecules embedded in gel were incubated with a nicking enzyme. The sequence downstream of the nick is CACTG. b) A first incorporation reaction, including only labeled dUTP and the polymerase, gave rise to only a few dots, presumably due to incorporation at random nicks preceding a T. c) An additional round of incorporation, including labeled dUTP and natural dCTP and dATP, was carried out. The labeled nucleotides were incorporated and appeared as fluorescent dots distributed along the length of the molecules.
    Figure Legend Snippet: Selective incorporation of labeled nucleotides. a) DNA molecules embedded in gel were incubated with a nicking enzyme. The sequence downstream of the nick is CACTG. b) A first incorporation reaction, including only labeled dUTP and the polymerase, gave rise to only a few dots, presumably due to incorporation at random nicks preceding a T. c) An additional round of incorporation, including labeled dUTP and natural dCTP and dATP, was carried out. The labeled nucleotides were incorporated and appeared as fluorescent dots distributed along the length of the molecules.

    Techniques Used: Labeling, Incubation, Sequencing

    DNA bridges are enzymatically active. a) DNA molecules embedded in a gel and stained with the intercalating dye YOYO-1 were visualized and are rendered in green. b) A first incorporation reaction including polymerase, Alexa 647-labeled dUTP and natural dCTP, dATP and dGTP was carried out. An image of the sample was captured in the Alexa 647 channel. Only a few dots appeared, most likely due to elongation from random nicks. c) After incubation with a nicking enzyme, the previous incorporation reaction was repeated. This time, many labeled nucleotides were incorporated and could be observed along the molecules.
    Figure Legend Snippet: DNA bridges are enzymatically active. a) DNA molecules embedded in a gel and stained with the intercalating dye YOYO-1 were visualized and are rendered in green. b) A first incorporation reaction including polymerase, Alexa 647-labeled dUTP and natural dCTP, dATP and dGTP was carried out. An image of the sample was captured in the Alexa 647 channel. Only a few dots appeared, most likely due to elongation from random nicks. c) After incubation with a nicking enzyme, the previous incorporation reaction was repeated. This time, many labeled nucleotides were incorporated and could be observed along the molecules.

    Techniques Used: Staining, Labeling, Incubation

    10) Product Images from "Dendritic cell combination therapy reduces the toxicity of triptolide and ameliorates colitis in murine models"

    Article Title: Dendritic cell combination therapy reduces the toxicity of triptolide and ameliorates colitis in murine models

    Journal: Drug Delivery

    doi: 10.1080/10717544.2022.2044935

    DCTP tracking in colitis mice. DCTP cells were labeled with DID and tracked by frozen sections, DAPI staining and fluorescence microscope observation. (A) DCTP was found in mLN and spleen but not colon. (B) No obvious DCTP was found in liver, kidney, heart, and testis (the scale bar represents 100 µm). Experiments were repeated three times.
    Figure Legend Snippet: DCTP tracking in colitis mice. DCTP cells were labeled with DID and tracked by frozen sections, DAPI staining and fluorescence microscope observation. (A) DCTP was found in mLN and spleen but not colon. (B) No obvious DCTP was found in liver, kidney, heart, and testis (the scale bar represents 100 µm). Experiments were repeated three times.

    Techniques Used: Mouse Assay, Labeling, Staining, Fluorescence, Microscopy

    11) Product Images from "IMP/GTP balance modulates cytoophidium assembly and IMPDH activity"

    Article Title: IMP/GTP balance modulates cytoophidium assembly and IMPDH activity

    Journal: Cell Division

    doi: 10.1186/s13008-018-0038-0

    IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments
    Figure Legend Snippet: IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments

    Techniques Used:

    12) Product Images from "A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection"

    Article Title: A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

    Journal: bioRxiv

    doi: 10.1101/2021.03.31.437871

    DcdV alters cellular nucleotide metabolism. ( A ) Lysates collected from E. coli expressing DcdV or DcdV E384A and a “no lysate” buffer control incubated with 12 nucleotide substrates (1.9 mM NH 4 Cl as a positive control, 37.7 mM cytidine, and 7.5 mM for all other substrates). Data represent the mean ± SEM, n =3. Quantification of dUTP ( B ) and dUMP ( C ) using UPLC-MS/MS, in the indicated cell lysates before (Pre) and after addition of 1 mM dCTP. Each lysate was normalized to 20 mg/mL total protein. Each bar represents mean ± SEM, n =3. ( D ) Quantification of the indicated dNTPs in vivo using UPLC-MS/MS in strains expressing the four DcdV variants, as indicated, normalized to dNTP concentrations measured in a vector control. Data are graphed as mean ± SEM, n= 3, Two-way ANOVA with Tukey’s multiple-comparison test, normalized to pVector, n.d. indicates “none detected”, and ns indicates “not significant”.
    Figure Legend Snippet: DcdV alters cellular nucleotide metabolism. ( A ) Lysates collected from E. coli expressing DcdV or DcdV E384A and a “no lysate” buffer control incubated with 12 nucleotide substrates (1.9 mM NH 4 Cl as a positive control, 37.7 mM cytidine, and 7.5 mM for all other substrates). Data represent the mean ± SEM, n =3. Quantification of dUTP ( B ) and dUMP ( C ) using UPLC-MS/MS, in the indicated cell lysates before (Pre) and after addition of 1 mM dCTP. Each lysate was normalized to 20 mg/mL total protein. Each bar represents mean ± SEM, n =3. ( D ) Quantification of the indicated dNTPs in vivo using UPLC-MS/MS in strains expressing the four DcdV variants, as indicated, normalized to dNTP concentrations measured in a vector control. Data are graphed as mean ± SEM, n= 3, Two-way ANOVA with Tukey’s multiple-comparison test, normalized to pVector, n.d. indicates “none detected”, and ns indicates “not significant”.

    Techniques Used: Expressing, Incubation, Positive Control, Tandem Mass Spectroscopy, In Vivo, Plasmid Preparation

    Addition of exogenous dTTP does not inhibit DcdV deaminase activity in E. coli lysates. Lysates collected from E. coli expressing WT DcdV incubated with or without exogenous 7.5 mM dTTP and either 75 mM cytidine, 7.5 mM dCMP, or 7.5 mM dCTP. The evolution of NH 4 + resulting from substrate deamination was detected by measuring the solution ABS 630 after a Berthelot’s reaction in microtiter plates. The relative deaminase activity was calculated by dividing the ABS 630 of the +dTTP reaction by the no dTTP control reaction for each lysate. Data represent the mean ± SEM of three biological replicate lysates.
    Figure Legend Snippet: Addition of exogenous dTTP does not inhibit DcdV deaminase activity in E. coli lysates. Lysates collected from E. coli expressing WT DcdV incubated with or without exogenous 7.5 mM dTTP and either 75 mM cytidine, 7.5 mM dCMP, or 7.5 mM dCTP. The evolution of NH 4 + resulting from substrate deamination was detected by measuring the solution ABS 630 after a Berthelot’s reaction in microtiter plates. The relative deaminase activity was calculated by dividing the ABS 630 of the +dTTP reaction by the no dTTP control reaction for each lysate. Data represent the mean ± SEM of three biological replicate lysates.

    Techniques Used: Activity Assay, Expressing, Incubation

    13) Product Images from "In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase"

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    Journal: Journal of Virology

    doi: 10.1128/JVI.07137-11

    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
    Figure Legend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Techniques Used: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

    Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).
    Figure Legend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Techniques Used: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

    Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.
    Figure Legend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

    14) Product Images from "Construction of an ~700-kb Transcript Map Around the Familial Mediterranean Fever Locus on Human Chromosome 16p13.3"

    Article Title: Construction of an ~700-kb Transcript Map Around the Familial Mediterranean Fever Locus on Human Chromosome 16p13.3

    Journal: Genome Research

    doi:

    Northern blot analysis of four transcripts from the FMF interval. Autoradiograms of human multitissue Northern blots probed with [ 32 P]dCTP-labeled cDNA are shown. Blots were probed with the following cDNAs: ( A ) clone CR17-26; ( B ) V9; ( C ) CR18-9; ( D ) ZNF174 .
    Figure Legend Snippet: Northern blot analysis of four transcripts from the FMF interval. Autoradiograms of human multitissue Northern blots probed with [ 32 P]dCTP-labeled cDNA are shown. Blots were probed with the following cDNAs: ( A ) clone CR17-26; ( B ) V9; ( C ) CR18-9; ( D ) ZNF174 .

    Techniques Used: Northern Blot, Labeling

    15) Product Images from "Inhibition of Polyprotein Processing and RNA Replication of Human Rhinovirus by Pyrrolidine Dithiocarbamate Involves Metal Ions"

    Article Title: Inhibition of Polyprotein Processing and RNA Replication of Human Rhinovirus by Pyrrolidine Dithiocarbamate Involves Metal Ions

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.22.13892-13899.2005

    PDTC decreases the replication of positive and negative strand RNA of HRV2. HeLa cells were infected with HRV2 (multiplicity of infection, 20). At the start of viral RNA replication at 4 h p.i. PDTC was added where indicated (+). Total RNA was isolated 1, 2, 4, 6, 8, and 13 h p.i. The level of positive- and negative-strand HRV2 RNA was quantitated by Northern blot analysis using strand-specific probes generated by primer extension in the presence of [γ- 32 P]dCTP and visualized by autoradiography. Analysis of the 28S rRNA ensured an equal loading.
    Figure Legend Snippet: PDTC decreases the replication of positive and negative strand RNA of HRV2. HeLa cells were infected with HRV2 (multiplicity of infection, 20). At the start of viral RNA replication at 4 h p.i. PDTC was added where indicated (+). Total RNA was isolated 1, 2, 4, 6, 8, and 13 h p.i. The level of positive- and negative-strand HRV2 RNA was quantitated by Northern blot analysis using strand-specific probes generated by primer extension in the presence of [γ- 32 P]dCTP and visualized by autoradiography. Analysis of the 28S rRNA ensured an equal loading.

    Techniques Used: Infection, Isolation, Northern Blot, Generated, Autoradiography

    16) Product Images from "The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site *"

    Article Title: The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.617720

    dTTP bound at the allosteric binding site. A , cross-eyed stereo view of an F o − F c map ( blue mesh ) at 3 σ calculated with dTTP omitted from the S-TIM5-T structure. Trp 42 is displayed as sticks, and the Mg 2+ ion is a yellow sphere. B , structural alignment of the allosteric binding sites of S-TIM5-T ( magenta ) and 2HVW ( yellow ). Trp 42 and dCMP from the S-TIM5-C structure are superimposed in cyan . The aromatic residue of the allosteric binding site and the bound nucleotide are represented as sticks. C , polar interactions made between dTTP ( green ) and the protein environment ( magenta ) are shown in black dashed lines . Interacting residues are labeled and represented with sticks , and the Mg 2+ ion is a yellow sphere. D , MST measurements. S-TIM5-dCD binding affinities to dCTP and dTTP. Standard deviations were obtained from triplicate experiments. Δ F Norm , normalized fluorescence units calculated as percentage of bound and unbound substrate.
    Figure Legend Snippet: dTTP bound at the allosteric binding site. A , cross-eyed stereo view of an F o − F c map ( blue mesh ) at 3 σ calculated with dTTP omitted from the S-TIM5-T structure. Trp 42 is displayed as sticks, and the Mg 2+ ion is a yellow sphere. B , structural alignment of the allosteric binding sites of S-TIM5-T ( magenta ) and 2HVW ( yellow ). Trp 42 and dCMP from the S-TIM5-C structure are superimposed in cyan . The aromatic residue of the allosteric binding site and the bound nucleotide are represented as sticks. C , polar interactions made between dTTP ( green ) and the protein environment ( magenta ) are shown in black dashed lines . Interacting residues are labeled and represented with sticks , and the Mg 2+ ion is a yellow sphere. D , MST measurements. S-TIM5-dCD binding affinities to dCTP and dTTP. Standard deviations were obtained from triplicate experiments. Δ F Norm , normalized fluorescence units calculated as percentage of bound and unbound substrate.

    Techniques Used: Binding Assay, Labeling, Microscale Thermophoresis, Fluorescence

    17) Product Images from "IMP/GTP balance modulates cytoophidium assembly and IMPDH activity"

    Article Title: IMP/GTP balance modulates cytoophidium assembly and IMPDH activity

    Journal: Cell Division

    doi: 10.1186/s13008-018-0038-0

    IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments
    Figure Legend Snippet: IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments

    Techniques Used:

    18) Product Images from "Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex"

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    Journal: bioRxiv

    doi: 10.1101/344556

    Adenine Mutagenesis and Template-Priming. (A) Covalently-linked RNA-cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. (B) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (-), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. (C) Percentage of substitutions in TR -cDNA determined by sequencing. (D) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by “+”,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (-G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. (E) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. (F) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase-and UDG-treated (bottom), and resolved by denaturing PAGE
    Figure Legend Snippet: Adenine Mutagenesis and Template-Priming. (A) Covalently-linked RNA-cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. (B) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (-), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. (C) Percentage of substitutions in TR -cDNA determined by sequencing. (D) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by “+”,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (-G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. (E) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. (F) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase-and UDG-treated (bottom), and resolved by denaturing PAGE

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    19) Product Images from "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures"

    Article Title: Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0710982105

    Principle of observing DNA synthesis inside ZMWs. ( A ) Template design. The minicircle DNA template contained a single guanine site, allowing incorporation of a base-linked fluorescent nucleotide, Alexa Fluor 488-dCTP. Rolling circle, DNA strand displacement
    Figure Legend Snippet: Principle of observing DNA synthesis inside ZMWs. ( A ) Template design. The minicircle DNA template contained a single guanine site, allowing incorporation of a base-linked fluorescent nucleotide, Alexa Fluor 488-dCTP. Rolling circle, DNA strand displacement

    Techniques Used: DNA Synthesis

    20) Product Images from "A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells"

    Article Title: A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells

    Journal: Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology

    doi: 10.1007/s10577-013-9358-8

    Effects of BrdU treatment on cell viability. a The bar graph shows colony formation assays of wt JM8.N4 ESCs to test effects of BrdU and BrdU/dCTP combinations at indicated concentrations. BrdU/dCTP concentrations are shown in micromolars. Cells were plated at equal densities on 6-well dishes (day 0), and after 1 day (day 1), were exposed to BrdU, dCTP, or BrdU/dCTP. Treatment time was 7 h, except for 40-μM BrdU (16 h). dCTP exposure was kept for another 24 h and was subsequently reduced in 50 % increments for two more days. On day 2, cells were trypsinized and split either 1:65,000 or 1:100,000, and colonies were counted after 1 week. Colony numbers were normalized to untreated control samples. Averages of three experiments are shown including error bars for standard deviations, except for one sample (1.25/0 μM BrdU/dCTP, indicated by an asterisk ) where the experiments were only carried out twice. b In one experiment, cells were split into 6-cm plates on day 2, and 7 days later colonies were stained for alkaline phosphatase ( AP ) activity. All colonies were AP positive
    Figure Legend Snippet: Effects of BrdU treatment on cell viability. a The bar graph shows colony formation assays of wt JM8.N4 ESCs to test effects of BrdU and BrdU/dCTP combinations at indicated concentrations. BrdU/dCTP concentrations are shown in micromolars. Cells were plated at equal densities on 6-well dishes (day 0), and after 1 day (day 1), were exposed to BrdU, dCTP, or BrdU/dCTP. Treatment time was 7 h, except for 40-μM BrdU (16 h). dCTP exposure was kept for another 24 h and was subsequently reduced in 50 % increments for two more days. On day 2, cells were trypsinized and split either 1:65,000 or 1:100,000, and colonies were counted after 1 week. Colony numbers were normalized to untreated control samples. Averages of three experiments are shown including error bars for standard deviations, except for one sample (1.25/0 μM BrdU/dCTP, indicated by an asterisk ) where the experiments were only carried out twice. b In one experiment, cells were split into 6-cm plates on day 2, and 7 days later colonies were stained for alkaline phosphatase ( AP ) activity. All colonies were AP positive

    Techniques Used: Staining, Activity Assay

    21) Product Images from "Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis"

    Article Title: Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis

    Journal: The Plant Cell

    doi:

    cDNA Microarray Analysis of Gene Expression after Mechanical Wounding. A fluorescently labeled cDNA probe was prepared from mRNA isolated from control Arabidopsis leaves by reverse transcription in the presence of Cy3-dCTP. A second probe, labeled with Cy5-dCTP, was prepared from leaves that were mechanically wounded (60 min). After the simultaneous hybridization of both probes with a cDNA microarray containing 150 defense-related Arabidopsis ESTs and scanning of the array, a pseudocolor image was generated. Genes induced or repressed after mechanical wounding are represented as red or green signals, respectively. Genes expressed at approximately equal levels between treatments appear as yellow spots. The intensity of each spot corresponds to the absolute amount of expression of each gene. The actual size of the array is 8 × 8 mm. Control genes are in the first row of top left, top right, and bottom left quadrants.
    Figure Legend Snippet: cDNA Microarray Analysis of Gene Expression after Mechanical Wounding. A fluorescently labeled cDNA probe was prepared from mRNA isolated from control Arabidopsis leaves by reverse transcription in the presence of Cy3-dCTP. A second probe, labeled with Cy5-dCTP, was prepared from leaves that were mechanically wounded (60 min). After the simultaneous hybridization of both probes with a cDNA microarray containing 150 defense-related Arabidopsis ESTs and scanning of the array, a pseudocolor image was generated. Genes induced or repressed after mechanical wounding are represented as red or green signals, respectively. Genes expressed at approximately equal levels between treatments appear as yellow spots. The intensity of each spot corresponds to the absolute amount of expression of each gene. The actual size of the array is 8 × 8 mm. Control genes are in the first row of top left, top right, and bottom left quadrants.

    Techniques Used: Microarray, Expressing, Labeling, Isolation, Hybridization, Generated

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    Thermo Fisher dctp
    The allosteric sites of SAMHD1 are highly restrictive. ( A ) Size-exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM <t>GTP</t> and 4 mM of color-coded nucleotide analog. ( B ) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 μM. ( C ) Malachite green activity assay performed in the presence of 125 μM GTP and 125 μM <t>dCTP,</t> dATP, nucleotide analog, or buffer. Error bars represent SEM of three independent experiments. ( D ) Malachite green activity assay measuring the hydrolysis of dATP by SAMHD1 tetramers preassembled in the presence of 125 μM GTP and 6.3 to 3,200 μM dATP, cladribine-TP, clofarabine-TP, or ATP. Error bars represent SEM of three independent experiments.
    Dctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa fluor 488 dctp
    Principle of observing DNA synthesis inside ZMWs. ( A ) Template design. The minicircle DNA template contained a single guanine site, allowing incorporation of a base-linked fluorescent nucleotide, <t>Alexa</t> Fluor <t>488-dCTP.</t> Rolling circle, DNA strand displacement
    Alexa Fluor 488 Dctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher alexa fluorescence conjugated secondary abs
    CpG-ODN induces the co-localization of DNA-PKcs with <t>TRAF6.</t> WT, DNA-PKcs −/− and TLR9 −/− DCs were seeded in an 8-well chamber slide, and then treated with CpG (5 µg/ml) for the indicated time points. The cells were fixed, permeabilized and immunostained with anti-DNA-PKcs Ab <t>(primary)/Alexa</t> 488 (2 nd Ab) (green) and anti-TRAF6 Ab (primary)/Alexa 594 (2 nd Ab) (red). Yellow color indicates co-localization of DNA-PKcs and TRAF6. The cells were observed under an IX81 Olympus microscope with 60× oil objective powered by 1.6× magnification. The images were recorded by an ORCA R2 CCD mono camera and analyzed by the Metamorph advanced for imaging software. Similar results were obtained from at least three independent experiments.
    Alexa Fluorescence Conjugated Secondary Abs, supplied by Thermo Fisher, 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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    Image Search Results


    The allosteric sites of SAMHD1 are highly restrictive. ( A ) Size-exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM GTP and 4 mM of color-coded nucleotide analog. ( B ) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 μM. ( C ) Malachite green activity assay performed in the presence of 125 μM GTP and 125 μM dCTP, dATP, nucleotide analog, or buffer. Error bars represent SEM of three independent experiments. ( D ) Malachite green activity assay measuring the hydrolysis of dATP by SAMHD1 tetramers preassembled in the presence of 125 μM GTP and 6.3 to 3,200 μM dATP, cladribine-TP, clofarabine-TP, or ATP. Error bars represent SEM of three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

    doi: 10.1073/pnas.1805593115

    Figure Lengend Snippet: The allosteric sites of SAMHD1 are highly restrictive. ( A ) Size-exclusion chromatography elution profile of SAMHD1 in the presence of 0.5 mM GTP and 4 mM of color-coded nucleotide analog. ( B ) SV-AUC analysis of SAMHD1 in the absence of nucleotides or the presence of dGTP, GTP with clofarabine-TP, or GTP with cladribine-TP at a final concentration of 150 μM. ( C ) Malachite green activity assay performed in the presence of 125 μM GTP and 125 μM dCTP, dATP, nucleotide analog, or buffer. Error bars represent SEM of three independent experiments. ( D ) Malachite green activity assay measuring the hydrolysis of dATP by SAMHD1 tetramers preassembled in the presence of 125 μM GTP and 6.3 to 3,200 μM dATP, cladribine-TP, clofarabine-TP, or ATP. Error bars represent SEM of three independent experiments.

    Article Snippet: GTP, dATP, and dCTP were purchased from Thermo Scientific.

    Techniques: Size-exclusion Chromatography, Concentration Assay, Activity Assay

    Role of Y374 and C2' sugar moiety substitution in acting as substrates of SAMHD1. A) dCTP, C) ara-CTP and E) SMDU-TP nucleotides (in green) are modeled within the catalytic site of SAMHD1. Both dCTP and ara-CTP do not clash with Y374 (see arrow). However the model shows that the (2' S )-2'-methyl group of SMDU-TP clashes with Y374 in the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, ara-CTP and SMDU-TP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs with experimental conditions described in Fig 2 . Data are presented as the percent compound remaining (y-axis). dCTP and ara-CTP are significantly hydrolyzed (p

    Journal: PLoS ONE

    Article Title: Substrates and Inhibitors of SAMHD1

    doi: 10.1371/journal.pone.0169052

    Figure Lengend Snippet: Role of Y374 and C2' sugar moiety substitution in acting as substrates of SAMHD1. A) dCTP, C) ara-CTP and E) SMDU-TP nucleotides (in green) are modeled within the catalytic site of SAMHD1. Both dCTP and ara-CTP do not clash with Y374 (see arrow). However the model shows that the (2' S )-2'-methyl group of SMDU-TP clashes with Y374 in the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, ara-CTP and SMDU-TP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs with experimental conditions described in Fig 2 . Data are presented as the percent compound remaining (y-axis). dCTP and ara-CTP are significantly hydrolyzed (p

    Article Snippet: Gemcitabine-5'-triphosphate (gem-TP), ara-cytidine-5'-triphosphate (ara-CTP) and 5-aza-2'-deoxycytidine-5'-triphosphate (decitabine-TP) were purchased from Jena Bioscience. dGTP, dATP and dCTP was purchased from Affymetrix.

    Techniques: Acetylene Reduction Assay, In Vitro

    Monitoring ara-CTP and gem-TP concentrations in MDMs. A) Monocyte-derived macrophages (MDMs) were pretreated with virus-like particles (VLP) one day prior to replacing the medium with fresh medium plus compounds: 10 μM of ara-C (cytaribine- 13 C 3 ) or 10 μM of gemcitabine. Whole cell lysates were collected at 0, 24 and 48 h post VLP addition. Lysates were analyzed by immunoblotting for SAMHD1 and GAPDH, loading control. SAMHD1 protein levels were reduced at 24 h after Vpx+ VLP exposure. Two human primary MDM donors are shown. Cellular nucleotide extracts were generated at 4, 12 and 24 h post drug addition from treated MDMs. The intracellular concentrations of B) gem-TP (2',2'-diF-dCTP) and (C) ara-CTP were quantified from the extracts using HPLC-MS/MS analysis. Data are plotted as pmol/million cells (y-axis) vs . time (h) (x-axis). Gem-TP is a significantly lower (**, p

    Journal: PLoS ONE

    Article Title: Substrates and Inhibitors of SAMHD1

    doi: 10.1371/journal.pone.0169052

    Figure Lengend Snippet: Monitoring ara-CTP and gem-TP concentrations in MDMs. A) Monocyte-derived macrophages (MDMs) were pretreated with virus-like particles (VLP) one day prior to replacing the medium with fresh medium plus compounds: 10 μM of ara-C (cytaribine- 13 C 3 ) or 10 μM of gemcitabine. Whole cell lysates were collected at 0, 24 and 48 h post VLP addition. Lysates were analyzed by immunoblotting for SAMHD1 and GAPDH, loading control. SAMHD1 protein levels were reduced at 24 h after Vpx+ VLP exposure. Two human primary MDM donors are shown. Cellular nucleotide extracts were generated at 4, 12 and 24 h post drug addition from treated MDMs. The intracellular concentrations of B) gem-TP (2',2'-diF-dCTP) and (C) ara-CTP were quantified from the extracts using HPLC-MS/MS analysis. Data are plotted as pmol/million cells (y-axis) vs . time (h) (x-axis). Gem-TP is a significantly lower (**, p

    Article Snippet: Gemcitabine-5'-triphosphate (gem-TP), ara-cytidine-5'-triphosphate (ara-CTP) and 5-aza-2'-deoxycytidine-5'-triphosphate (decitabine-TP) were purchased from Jena Bioscience. dGTP, dATP and dCTP was purchased from Affymetrix.

    Techniques: Acetylene Reduction Assay, Derivative Assay, Generated, High Performance Liquid Chromatography, Mass Spectrometry

    Ara-CTP does not fit into the A2 site of SAMHD1. A) Evaluating ara-CTP hydrolysis in the presence of dGTP, using as A1site activator. When dGTP was present, ara-CTP and dCTP were significantly hydrolyzed (p

    Journal: PLoS ONE

    Article Title: Substrates and Inhibitors of SAMHD1

    doi: 10.1371/journal.pone.0169052

    Figure Lengend Snippet: Ara-CTP does not fit into the A2 site of SAMHD1. A) Evaluating ara-CTP hydrolysis in the presence of dGTP, using as A1site activator. When dGTP was present, ara-CTP and dCTP were significantly hydrolyzed (p

    Article Snippet: Gemcitabine-5'-triphosphate (gem-TP), ara-cytidine-5'-triphosphate (ara-CTP) and 5-aza-2'-deoxycytidine-5'-triphosphate (decitabine-TP) were purchased from Jena Bioscience. dGTP, dATP and dCTP was purchased from Affymetrix.

    Techniques: Acetylene Reduction Assay

    Examining the role of L150 for nucleotide specificity. A) dCTP, C) (2' R ) 2'-F-dCTP and E) CTP nucleotides (in green) are modeled in the catalytic site of SAMHD1. L150 clashes with (2' R ) 2'-F-dCTP and CTP, but not dCTP (see arrows) within the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, (2' R ) 2'-F-dCTP and CTP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs. Using semi-quantitative HLPC analysis method, compounds were incubated with and without 1.6 μM of SAMHD1 enzyme plus dGTP (A1 site activator) to determine if they are substrates of SAMHD1. Data are presented as the percent compound remaining (y-axis). dCTP and dGTP were significantly hydrolyzed (p

    Journal: PLoS ONE

    Article Title: Substrates and Inhibitors of SAMHD1

    doi: 10.1371/journal.pone.0169052

    Figure Lengend Snippet: Examining the role of L150 for nucleotide specificity. A) dCTP, C) (2' R ) 2'-F-dCTP and E) CTP nucleotides (in green) are modeled in the catalytic site of SAMHD1. L150 clashes with (2' R ) 2'-F-dCTP and CTP, but not dCTP (see arrows) within the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, (2' R ) 2'-F-dCTP and CTP can be hydrolyzed for SAMHD1 in vitro . Structures of the compounds are above the HLPC graphs. Using semi-quantitative HLPC analysis method, compounds were incubated with and without 1.6 μM of SAMHD1 enzyme plus dGTP (A1 site activator) to determine if they are substrates of SAMHD1. Data are presented as the percent compound remaining (y-axis). dCTP and dGTP were significantly hydrolyzed (p

    Article Snippet: Gemcitabine-5'-triphosphate (gem-TP), ara-cytidine-5'-triphosphate (ara-CTP) and 5-aza-2'-deoxycytidine-5'-triphosphate (decitabine-TP) were purchased from Jena Bioscience. dGTP, dATP and dCTP was purchased from Affymetrix.

    Techniques: In Vitro, Incubation

    Principle of observing DNA synthesis inside ZMWs. ( A ) Template design. The minicircle DNA template contained a single guanine site, allowing incorporation of a base-linked fluorescent nucleotide, Alexa Fluor 488-dCTP. Rolling circle, DNA strand displacement

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

    doi: 10.1073/pnas.0710982105

    Figure Lengend Snippet: Principle of observing DNA synthesis inside ZMWs. ( A ) Template design. The minicircle DNA template contained a single guanine site, allowing incorporation of a base-linked fluorescent nucleotide, Alexa Fluor 488-dCTP. Rolling circle, DNA strand displacement

    Article Snippet: For indirect DNA staining , Alexa Fluor 488-dCTP was replaced by dCTP, with an additional incubation step using SybrGold DNA stain at 1:102 dilution in buffer A for 10 min at room temperature, followed by 5× washing in buffer A (Invitrogen).

    Techniques: DNA Synthesis

    CpG-ODN induces the co-localization of DNA-PKcs with TRAF6. WT, DNA-PKcs −/− and TLR9 −/− DCs were seeded in an 8-well chamber slide, and then treated with CpG (5 µg/ml) for the indicated time points. The cells were fixed, permeabilized and immunostained with anti-DNA-PKcs Ab (primary)/Alexa 488 (2 nd Ab) (green) and anti-TRAF6 Ab (primary)/Alexa 594 (2 nd Ab) (red). Yellow color indicates co-localization of DNA-PKcs and TRAF6. The cells were observed under an IX81 Olympus microscope with 60× oil objective powered by 1.6× magnification. The images were recorded by an ORCA R2 CCD mono camera and analyzed by the Metamorph advanced for imaging software. Similar results were obtained from at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Involvement of DNA-PKcs in the IL-6 and IL-12 Response to CpG-ODN Is Mediated by Its Interaction with TRAF6 in Dendritic Cells

    doi: 10.1371/journal.pone.0058072

    Figure Lengend Snippet: CpG-ODN induces the co-localization of DNA-PKcs with TRAF6. WT, DNA-PKcs −/− and TLR9 −/− DCs were seeded in an 8-well chamber slide, and then treated with CpG (5 µg/ml) for the indicated time points. The cells were fixed, permeabilized and immunostained with anti-DNA-PKcs Ab (primary)/Alexa 488 (2 nd Ab) (green) and anti-TRAF6 Ab (primary)/Alexa 594 (2 nd Ab) (red). Yellow color indicates co-localization of DNA-PKcs and TRAF6. The cells were observed under an IX81 Olympus microscope with 60× oil objective powered by 1.6× magnification. The images were recorded by an ORCA R2 CCD mono camera and analyzed by the Metamorph advanced for imaging software. Similar results were obtained from at least three independent experiments.

    Article Snippet: Anti-TLR9 mouse monoclonal (mAb) was from Sigma; anti-DNA-PKcs mAb was from Becton Dickinson (BD) PharMingen (CA) or Neomarker (CA), and anti-DNA-PKcs polyclonal rabbit antibody was from Santa Cruz Biotech (CA); anti-TRAF6 Ab was from Santa Cruz Biotech; and Alexa fluorescence-conjugated secondary Abs were from Invitrogen.

    Techniques: Microscopy, Imaging, Software