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    Structured Review

    Thermo Fisher deoxynucleotide triphosphate
    Deoxynucleotide Triphosphate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxynucleotide triphosphate/product/Thermo Fisher
    Average 99 stars, based on 340 article reviews
    Price from $9.99 to $1999.99
    deoxynucleotide triphosphate - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Transduction:

    Article Title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs
    Article Snippet: .. THP-1 cells transduced with lentiviral vectors encoding SAMHD1 shRNA were selected by culturing in a medium containing 0.8 μg/ml puromycin. dNs consisted of a mixture of dA (D8668), dC (D0776), dG (D0901) and dT (T1895; all from Sigma-Aldrich). dNTPs (R0192) were purchased from Fermentas (Glen Burnie, MD). .. Cytokine cocktail for HSPC transduction consisted of a mixture of stem cell factor (SCF) at 100 ng/ml, Fms-related tyrosine kinase 3 ligand (Flt3L) at 100 ng/ml, interleukin-3 (IL-3) at 60 ng/ml and thrombopoietin (TPO) at 10 ng/ml (Immunotools, Friesoythe, Germany).

    Amplification:

    Article Title: Evolution of blue-flowered species of genus Linum based on high-throughput sequencing of ribosomal RNA genes
    Article Snippet: .. Amplification was carried out in 25 μl of PCR mix containing 1× KAPA2G buffer A (Kapa Biosystems), 250 μM each dNTP (Thermo Scientific, USA), 0.12 μM of MIDх-454 forward and reverse primers, 0.5 U of KAPA2G Fast HotStart DNA Polymerase (Kapa Biosystems), and 3 μl of the diluted (1:20) first-stage PCR product. .. The PCR products were separated in 1.5% agarose gel for 5 h at 45 V using ТВЕ buffer and then stained with ethidium bromide.

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Article Title: Significance of UGT1A1*28 Genotype in Patients with Advanced Liver Injury Caused By Chronic Hepatitis C
    Article Snippet: .. The amplification reaction was performed in a total volume of 25 μL, and the reaction mix contained 20 pmol of each primer, 50–100 ng of genomic DNA, 200 mmol/L of each dNTP (Fermentas, ON, Canada), 1× PCR reaction buffer (Qiagen), 1× Q solution (Qiagen), 2.75 mmol/L MgCl2 , 1 U HotStar DNA polymerase (Qiagen). .. The temperature profile of the PCR reactions was for the initial activation of DNA polymerase set at 95 °C for 15 min, followed by 35 cycles of 30 sec denaturation at 95 °C, 30 sec annealing at 63 °C, and 30 sec elongation at 72 °C, ended by a final extension period of 7 min at 72 °C.

    Synthesized:

    Article Title: Correlation between CXCR4/CXCR7/CXCL12 chemokine axis expression and prognosis in lymph‐node‐positive lung cancer patients, et al. Correlation between CXCR4/CXCR7/CXCL12 chemokine axis expression and prognosis in lymph‐node‐positive lung cancer patients
    Article Snippet: .. RNA was converted into cDNA using the Prime Script RT Master Mix (Takara Bio, Shiga, Japan) according to the manufacturer's protocol. cDNA was synthesized using 500 ng of each mRNA sample with Oligo‐dT‐Primers (Life Technologies, Darmstadt, Germany), Superscript II Reverse Transcriptase (Life Technologies) and dNTP (Fermentas, Waltham, MA, USA) following the manufacturer's instructions. .. The cDNA was diluted fivefold and used for real‐time PCR analysis with the Thermal Cycler Dice Real Time System II TP‐900 (Takara Bio) using a TaqMan Gene Expression Assay probe and primer mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    shRNA:

    Article Title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs
    Article Snippet: .. THP-1 cells transduced with lentiviral vectors encoding SAMHD1 shRNA were selected by culturing in a medium containing 0.8 μg/ml puromycin. dNs consisted of a mixture of dA (D8668), dC (D0776), dG (D0901) and dT (T1895; all from Sigma-Aldrich). dNTPs (R0192) were purchased from Fermentas (Glen Burnie, MD). .. Cytokine cocktail for HSPC transduction consisted of a mixture of stem cell factor (SCF) at 100 ng/ml, Fms-related tyrosine kinase 3 ligand (Flt3L) at 100 ng/ml, interleukin-3 (IL-3) at 60 ng/ml and thrombopoietin (TPO) at 10 ng/ml (Immunotools, Friesoythe, Germany).

    Polymerase Chain Reaction:

    Article Title: PCR detection of Burkholderia multivorans in water and soil samples
    Article Snippet: .. The PCR mix contained 1× CorelLoad PCR buffer (Qiagen), 0.25 mM dNTP (Applied Biosystems), 1 U Taq (Qiagen), 0.5 μM of both primers, 1× Q-solution (Qiagen) and 200 ng/μl BSA (Roche). .. For each sample, 2 μl DNA was added to 23 μl PCR master mix.

    Article Title: UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells
    Article Snippet: .. A common PCR mix for CrRPS9 and CrDHS1 for each treatment was prepared containing one μl of the RT reaction mix per 20 μl reaction volume containing 0.4 U of Taq DNA polymerase (Fermentas), 0.1 mM dNTP (Fermentas) and 200 μM of each dNTP and later split into equal half to add 100 pM of gene-specific primer in a 1X reaction buffer. .. Reactions were amplified for a total of 15 cycles in the Minicycler (MJ Research PTC-150) using 94°C denaturation (1 min), 52°C annealing for CrDHS1 and 50°C for CrRPS9 (1 min) and 72°C extension (1 min), followed by a further 5 min extension.

    Article Title: A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering
    Article Snippet: .. PCR with dUTP replacing dTTP was performed using standard Phusion (Finnzymes) reaction conditions as recommended by the manufacturer, using the dUTP/dNTPs mix (Fermentas) instead of dNTPs. .. The pET-Pfu-vector was a kind gift from Jens Lykke-Andersen.

    Article Title: Evolution of blue-flowered species of genus Linum based on high-throughput sequencing of ribosomal RNA genes
    Article Snippet: .. Amplification was carried out in 25 μl of PCR mix containing 1× KAPA2G buffer A (Kapa Biosystems), 250 μM each dNTP (Thermo Scientific, USA), 0.12 μM of MIDх-454 forward and reverse primers, 0.5 U of KAPA2G Fast HotStart DNA Polymerase (Kapa Biosystems), and 3 μl of the diluted (1:20) first-stage PCR product. .. The PCR products were separated in 1.5% agarose gel for 5 h at 45 V using ТВЕ buffer and then stained with ethidium bromide.

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Article Title: Significance of UGT1A1*28 Genotype in Patients with Advanced Liver Injury Caused By Chronic Hepatitis C
    Article Snippet: .. The amplification reaction was performed in a total volume of 25 μL, and the reaction mix contained 20 pmol of each primer, 50–100 ng of genomic DNA, 200 mmol/L of each dNTP (Fermentas, ON, Canada), 1× PCR reaction buffer (Qiagen), 1× Q solution (Qiagen), 2.75 mmol/L MgCl2 , 1 U HotStar DNA polymerase (Qiagen). .. The temperature profile of the PCR reactions was for the initial activation of DNA polymerase set at 95 °C for 15 min, followed by 35 cycles of 30 sec denaturation at 95 °C, 30 sec annealing at 63 °C, and 30 sec elongation at 72 °C, ended by a final extension period of 7 min at 72 °C.

    Staining:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Plasmid Preparation:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

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  • 94
    Thermo Fisher gene exp samhd1 hs00210019 m1
    Proposed model for p21-mediated HIV-1 restriction in MDDCs. The expression of p21 is induced during MDDC maturation (step 1). The increase in p21 expression leads to the downregulation of several enzymes (RNR2, TYMS, and TK1) involved in dNTP biosynthesis (step 2), which would decrease the dNTP pool size (step 3), impairing HIV-1 reverse transcription (step 4). In parallel, p21 induction could increase the active isoform of <t>SAMHD1</t> (step 5), which would further block viral replication through the degradation of dNTPs (step 6) or exonuclease activity (step 7). Low levels of dNTP synthesis as a consequence of p21 induction might further facilitate the exonuclease activity of SAMHD1 (step 8). Although not explored here, induction of p21 might also directly block reverse transcriptase activity and impair HIV transcription, as has been shown in CD4 + ).
    Gene Exp Samhd1 Hs00210019 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp samhd1 hs00210019 m1/product/Thermo Fisher
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    gene exp samhd1 hs00210019 m1 - by Bioz Stars, 2020-07
    94/100 stars
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    92
    Thermo Fisher deoxynucleotide triphosphate
    Proposed model for p21-mediated HIV-1 restriction in MDDCs. The expression of p21 is induced during MDDC maturation (step 1). The increase in p21 expression leads to the downregulation of several enzymes (RNR2, TYMS, and TK1) involved in dNTP biosynthesis (step 2), which would decrease the dNTP pool size (step 3), impairing HIV-1 reverse transcription (step 4). In parallel, p21 induction could increase the active isoform of <t>SAMHD1</t> (step 5), which would further block viral replication through the degradation of dNTPs (step 6) or exonuclease activity (step 7). Low levels of dNTP synthesis as a consequence of p21 induction might further facilitate the exonuclease activity of SAMHD1 (step 8). Although not explored here, induction of p21 might also directly block reverse transcriptase activity and impair HIV transcription, as has been shown in CD4 + ).
    Deoxynucleotide Triphosphate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxynucleotide triphosphate/product/Thermo Fisher
    Average 92 stars, based on 340 article reviews
    Price from $9.99 to $1999.99
    deoxynucleotide triphosphate - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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    Proposed model for p21-mediated HIV-1 restriction in MDDCs. The expression of p21 is induced during MDDC maturation (step 1). The increase in p21 expression leads to the downregulation of several enzymes (RNR2, TYMS, and TK1) involved in dNTP biosynthesis (step 2), which would decrease the dNTP pool size (step 3), impairing HIV-1 reverse transcription (step 4). In parallel, p21 induction could increase the active isoform of SAMHD1 (step 5), which would further block viral replication through the degradation of dNTPs (step 6) or exonuclease activity (step 7). Low levels of dNTP synthesis as a consequence of p21 induction might further facilitate the exonuclease activity of SAMHD1 (step 8). Although not explored here, induction of p21 might also directly block reverse transcriptase activity and impair HIV transcription, as has been shown in CD4 + ).

    Journal: Journal of Virology

    Article Title: p21 Restricts HIV-1 in Monocyte-Derived Dendritic Cells through the Reduction of Deoxynucleoside Triphosphate Biosynthesis and Regulation of SAMHD1 Antiviral Activity

    doi: 10.1128/JVI.01324-17

    Figure Lengend Snippet: Proposed model for p21-mediated HIV-1 restriction in MDDCs. The expression of p21 is induced during MDDC maturation (step 1). The increase in p21 expression leads to the downregulation of several enzymes (RNR2, TYMS, and TK1) involved in dNTP biosynthesis (step 2), which would decrease the dNTP pool size (step 3), impairing HIV-1 reverse transcription (step 4). In parallel, p21 induction could increase the active isoform of SAMHD1 (step 5), which would further block viral replication through the degradation of dNTPs (step 6) or exonuclease activity (step 7). Low levels of dNTP synthesis as a consequence of p21 induction might further facilitate the exonuclease activity of SAMHD1 (step 8). Although not explored here, induction of p21 might also directly block reverse transcriptase activity and impair HIV transcription, as has been shown in CD4 + ).

    Article Snippet: PCR amplification of cDNAs was carried out as previously described ( ) on a 7500 real-time PCR system using the TaqMan gene expression premade mix (Applied Biosystems) to amplify the following genes: RRM2 (Hs00357247_g1), TYMS (Hs00426586_m1), SAMHD1 (Hs00210019_m1), CTPS1 (Hs01041858_m1), TK1 (Hs01062125_m1), CDKN1A (Hs00355782_m1), and GAPDH (Hs02758991_g1).

    Techniques: Expressing, Blocking Assay, Activity Assay

    p21 expression modulates SAMHD1 phosphorylation levels in MDDCs. (A to C) Relative copies of SAMHD1 mRNA (A), total SAMHD1 protein levels (B), and pSAMHD1 protein levels (C) in immature and mature MDDC after transfection with siRNA p21 or siRNA NT. The data are expressed relative to the number of mRNA copies or protein levels in immature MDDCs transfected with nontargeting siRNA. The symbols represent independent experiments ( n = 9 donors for mRNA and n = 4 donors for proteins). (D) Changes in luciferase activity after infection with HIV-1 NL4.3ΔenvLuc VSV-G in immature and mature MDDCs cultured in the presence of virus-like particles with or without the SIV VPX protein. The results are shown as the fold change relative to the luciferase activity in immature MDDCs cultured with VLPs not carrying VPX. The symbols represent independent experiments ( n = 9). The median and IQR values are shown. P values of ANOVA are shown. Significant differences ( P

    Journal: Journal of Virology

    Article Title: p21 Restricts HIV-1 in Monocyte-Derived Dendritic Cells through the Reduction of Deoxynucleoside Triphosphate Biosynthesis and Regulation of SAMHD1 Antiviral Activity

    doi: 10.1128/JVI.01324-17

    Figure Lengend Snippet: p21 expression modulates SAMHD1 phosphorylation levels in MDDCs. (A to C) Relative copies of SAMHD1 mRNA (A), total SAMHD1 protein levels (B), and pSAMHD1 protein levels (C) in immature and mature MDDC after transfection with siRNA p21 or siRNA NT. The data are expressed relative to the number of mRNA copies or protein levels in immature MDDCs transfected with nontargeting siRNA. The symbols represent independent experiments ( n = 9 donors for mRNA and n = 4 donors for proteins). (D) Changes in luciferase activity after infection with HIV-1 NL4.3ΔenvLuc VSV-G in immature and mature MDDCs cultured in the presence of virus-like particles with or without the SIV VPX protein. The results are shown as the fold change relative to the luciferase activity in immature MDDCs cultured with VLPs not carrying VPX. The symbols represent independent experiments ( n = 9). The median and IQR values are shown. P values of ANOVA are shown. Significant differences ( P

    Article Snippet: PCR amplification of cDNAs was carried out as previously described ( ) on a 7500 real-time PCR system using the TaqMan gene expression premade mix (Applied Biosystems) to amplify the following genes: RRM2 (Hs00357247_g1), TYMS (Hs00426586_m1), SAMHD1 (Hs00210019_m1), CTPS1 (Hs01041858_m1), TK1 (Hs01062125_m1), CDKN1A (Hs00355782_m1), and GAPDH (Hs02758991_g1).

    Techniques: Expressing, Transfection, Luciferase, Activity Assay, Infection, Cell Culture

    Phosphorylation state of SAMHD1. Monocytes (Monocyte), M-CSF-differentiated macrophages (Macrophage (M-CSF)), and GM-CSF-differentiated macrophages (Macrophage (GM-CSF)) were treated with GH123-Nhe/VSV-G for 2 h and incubated at 37°C for 4 h (4 h) or 24 h (24 h), or mock-treated (−). Cells were lyzed and subjected for SDS-PAGE containing Phos-tag to separate phosphorylated proteins from nonphosphorylated ones. SAMHD1 proteins were detected by anti-SAMHD1 antibody. Upper bands shown by a vertical bar and a lower band shown by an arrow represent phosphorylated and nonphosphorylated SAMHD1, respectively. Ratios of phosphorylated SAMHD1 levels to total SAMHD1 levels (ratio) are shown in vertical numbers.

    Journal: PLoS ONE

    Article Title: Moderate Restriction of Macrophage-Tropic Human Immunodeficiency Virus Type 1 by SAMHD1 in Monocyte-Derived Macrophages

    doi: 10.1371/journal.pone.0090969

    Figure Lengend Snippet: Phosphorylation state of SAMHD1. Monocytes (Monocyte), M-CSF-differentiated macrophages (Macrophage (M-CSF)), and GM-CSF-differentiated macrophages (Macrophage (GM-CSF)) were treated with GH123-Nhe/VSV-G for 2 h and incubated at 37°C for 4 h (4 h) or 24 h (24 h), or mock-treated (−). Cells were lyzed and subjected for SDS-PAGE containing Phos-tag to separate phosphorylated proteins from nonphosphorylated ones. SAMHD1 proteins were detected by anti-SAMHD1 antibody. Upper bands shown by a vertical bar and a lower band shown by an arrow represent phosphorylated and nonphosphorylated SAMHD1, respectively. Ratios of phosphorylated SAMHD1 levels to total SAMHD1 levels (ratio) are shown in vertical numbers.

    Article Snippet: For real time PCR, each 20-µL reaction mixture consisted of 5 µL of cDNA, 10 µL TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA) and 1 µL of TaqMan Gene Ex Assays (Assay ID: Hs00210019_m1).

    Techniques: Incubation, SDS Page

    Quantification of SAMHD1 mRNA expression. Monocytes were differentiated into macrophages for 6 days in the presence of GM-CSF or M-CSF. Macrophages or monocytes were treated with or without VSV-G-pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G), and harvested. SAMHD1 mRNA expression levels were detected by real-time RT-PCR and normalized against GAPDH. Data are shown as mean ± SD.

    Journal: PLoS ONE

    Article Title: Moderate Restriction of Macrophage-Tropic Human Immunodeficiency Virus Type 1 by SAMHD1 in Monocyte-Derived Macrophages

    doi: 10.1371/journal.pone.0090969

    Figure Lengend Snippet: Quantification of SAMHD1 mRNA expression. Monocytes were differentiated into macrophages for 6 days in the presence of GM-CSF or M-CSF. Macrophages or monocytes were treated with or without VSV-G-pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G), and harvested. SAMHD1 mRNA expression levels were detected by real-time RT-PCR and normalized against GAPDH. Data are shown as mean ± SD.

    Article Snippet: For real time PCR, each 20-µL reaction mixture consisted of 5 µL of cDNA, 10 µL TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA) and 1 µL of TaqMan Gene Ex Assays (Assay ID: Hs00210019_m1).

    Techniques: Expressing, Quantitative RT-PCR

    Effects of SAMHD1 siRNA on HIV-1 infection. Monocytes (Monocyte) or macrophages (Macrophage) were treated with SAMHD1- (siSAMHD1) or GFP- (siGFP) targeting siRNAs. Three days after transfection, cells were harvested or infected with NL43-Luci/VSV-G virus. SAMHD1 mRNA expression levels in harvested cells were detected by real-time RT-PCR and normalized against GAPDH. Data are shown as mean ± SD. Luciferase activity in infected cells was measured 4 days after infection. Data are plotted as the mean ± SD of triplicate samples; presented data are representative of two independent experiments using two donors. A: Results of samples obtained from a donor 1. B: Results of samples obtained from a donor 2.

    Journal: PLoS ONE

    Article Title: Moderate Restriction of Macrophage-Tropic Human Immunodeficiency Virus Type 1 by SAMHD1 in Monocyte-Derived Macrophages

    doi: 10.1371/journal.pone.0090969

    Figure Lengend Snippet: Effects of SAMHD1 siRNA on HIV-1 infection. Monocytes (Monocyte) or macrophages (Macrophage) were treated with SAMHD1- (siSAMHD1) or GFP- (siGFP) targeting siRNAs. Three days after transfection, cells were harvested or infected with NL43-Luci/VSV-G virus. SAMHD1 mRNA expression levels in harvested cells were detected by real-time RT-PCR and normalized against GAPDH. Data are shown as mean ± SD. Luciferase activity in infected cells was measured 4 days after infection. Data are plotted as the mean ± SD of triplicate samples; presented data are representative of two independent experiments using two donors. A: Results of samples obtained from a donor 1. B: Results of samples obtained from a donor 2.

    Article Snippet: For real time PCR, each 20-µL reaction mixture consisted of 5 µL of cDNA, 10 µL TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA) and 1 µL of TaqMan Gene Ex Assays (Assay ID: Hs00210019_m1).

    Techniques: Infection, Transfection, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay

    Western blot analysis of SAMHD1 in undifferentiated monocytes and macrophages. Monocytes were differentiated into macrophages for 6 days in the presence of GM-CSF or M-CSF. Macrophages or monocytes were treated with or without VSV-G-pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G), and harvested. Whole-cell extracts were separated on SDS-PAGE and analyzed by western blot using the indicated antibodies. Presented data are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Moderate Restriction of Macrophage-Tropic Human Immunodeficiency Virus Type 1 by SAMHD1 in Monocyte-Derived Macrophages

    doi: 10.1371/journal.pone.0090969

    Figure Lengend Snippet: Western blot analysis of SAMHD1 in undifferentiated monocytes and macrophages. Monocytes were differentiated into macrophages for 6 days in the presence of GM-CSF or M-CSF. Macrophages or monocytes were treated with or without VSV-G-pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G), and harvested. Whole-cell extracts were separated on SDS-PAGE and analyzed by western blot using the indicated antibodies. Presented data are representative of two independent experiments.

    Article Snippet: For real time PCR, each 20-µL reaction mixture consisted of 5 µL of cDNA, 10 µL TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA) and 1 µL of TaqMan Gene Ex Assays (Assay ID: Hs00210019_m1).

    Techniques: Western Blot, SDS Page

    Relationship of SAMHD1 transcription, Type I interferon response, and disease severity. There was a significant positive correlation of SAMHD1 transcript and level of MX1, an indicator of the Type I interferon response ( A ). Comparing Mx1 ( B ) and SAMHD1 ( C ) transcript with disease severity as assigned by pathology (none, mild, moderate, or severe), the association of Mx1 with disease appeared stronger than the association of SAMHD1 and disease. Transcript abundance was normalized to the average of beta actin and GAPDH; hence, the smaller the “dCq” value, the greater the abundance.

    Journal: Scientific Reports

    Article Title: SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load

    doi: 10.1038/srep22629

    Figure Lengend Snippet: Relationship of SAMHD1 transcription, Type I interferon response, and disease severity. There was a significant positive correlation of SAMHD1 transcript and level of MX1, an indicator of the Type I interferon response ( A ). Comparing Mx1 ( B ) and SAMHD1 ( C ) transcript with disease severity as assigned by pathology (none, mild, moderate, or severe), the association of Mx1 with disease appeared stronger than the association of SAMHD1 and disease. Transcript abundance was normalized to the average of beta actin and GAPDH; hence, the smaller the “dCq” value, the greater the abundance.

    Article Snippet: Pre-designed TaqMan Assays (Applied Biosystems/Life Technologies) were used to quantitate SAMHD1, Mx1, GAPDH, and ACTB from human (Hs00210019_m1, Hs00895608_m1, Hs02758991_g1, and Hs99999903_m1, respectively) and macaque (Rh02869978_m1, Rh02842279_m1, Rh02621745_g1, and Rh03043379_gH, respectively).

    Techniques:

    Brain SAMHD1 transcript load during SIV infection. SAMHD1 transcript increased in thalamus during SIV infection, with peak expression corresponding with acute and late-stage infection. SAMHD1 mRNA was quantitated by qPCR, with normalization to the average of beta actin and GAPDH. **p

    Journal: Scientific Reports

    Article Title: SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load

    doi: 10.1038/srep22629

    Figure Lengend Snippet: Brain SAMHD1 transcript load during SIV infection. SAMHD1 transcript increased in thalamus during SIV infection, with peak expression corresponding with acute and late-stage infection. SAMHD1 mRNA was quantitated by qPCR, with normalization to the average of beta actin and GAPDH. **p

    Article Snippet: Pre-designed TaqMan Assays (Applied Biosystems/Life Technologies) were used to quantitate SAMHD1, Mx1, GAPDH, and ACTB from human (Hs00210019_m1, Hs00895608_m1, Hs02758991_g1, and Hs99999903_m1, respectively) and macaque (Rh02869978_m1, Rh02842279_m1, Rh02621745_g1, and Rh03043379_gH, respectively).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction

    Viral load and SAMHD1. Tissue viral load as determined by standard curve-normalized qPCR did not correlate with SAMHD1 transcript (normalized to the average of beta actin and GAPDH).

    Journal: Scientific Reports

    Article Title: SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load

    doi: 10.1038/srep22629

    Figure Lengend Snippet: Viral load and SAMHD1. Tissue viral load as determined by standard curve-normalized qPCR did not correlate with SAMHD1 transcript (normalized to the average of beta actin and GAPDH).

    Article Snippet: Pre-designed TaqMan Assays (Applied Biosystems/Life Technologies) were used to quantitate SAMHD1, Mx1, GAPDH, and ACTB from human (Hs00210019_m1, Hs00895608_m1, Hs02758991_g1, and Hs99999903_m1, respectively) and macaque (Rh02869978_m1, Rh02842279_m1, Rh02621745_g1, and Rh03043379_gH, respectively).

    Techniques: Real-time Polymerase Chain Reaction

    SAMHD1 and IFNB1 in primary macrophages. M×1 of primary human monocyte-derived macrophages responded rapidly and strongly to 100 U/mL treatment with recombinant IFNB1, but not to TNFalpha (20 ng/mL, ( A )). SAMHD1 also responded to IFNB1, but the increase was detectable and significant only by 12 hours post-treatment ( B ). HIV-1 (BaL) infection of primary monocyte-derived macrophages yielded an apparent increase of SAMHD1 levels by 7 and 10 days post-infection, but this increase was not statistically significant (NS) ( C ). ***p

    Journal: Scientific Reports

    Article Title: SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load

    doi: 10.1038/srep22629

    Figure Lengend Snippet: SAMHD1 and IFNB1 in primary macrophages. M×1 of primary human monocyte-derived macrophages responded rapidly and strongly to 100 U/mL treatment with recombinant IFNB1, but not to TNFalpha (20 ng/mL, ( A )). SAMHD1 also responded to IFNB1, but the increase was detectable and significant only by 12 hours post-treatment ( B ). HIV-1 (BaL) infection of primary monocyte-derived macrophages yielded an apparent increase of SAMHD1 levels by 7 and 10 days post-infection, but this increase was not statistically significant (NS) ( C ). ***p

    Article Snippet: Pre-designed TaqMan Assays (Applied Biosystems/Life Technologies) were used to quantitate SAMHD1, Mx1, GAPDH, and ACTB from human (Hs00210019_m1, Hs00895608_m1, Hs02758991_g1, and Hs99999903_m1, respectively) and macaque (Rh02869978_m1, Rh02842279_m1, Rh02621745_g1, and Rh03043379_gH, respectively).

    Techniques: Derivative Assay, Recombinant, Infection

    Primary astrocyte SAMHD1 responds to SIV infection and IFNB1. Quantitative PCR results, normalized to the average of beta actin and GAPDH, indicated that SIV infection (SIV 17E-Fr) and IFNB1 treatment of primary rhesus astrocytes resulted in significant upregulation of SAMHD1 by 48 hours post infection and at all subsequent time points examined. SIV infection alone stimulated apparently significant upregulation at two and 14 days post infection. ***p

    Journal: Scientific Reports

    Article Title: SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load

    doi: 10.1038/srep22629

    Figure Lengend Snippet: Primary astrocyte SAMHD1 responds to SIV infection and IFNB1. Quantitative PCR results, normalized to the average of beta actin and GAPDH, indicated that SIV infection (SIV 17E-Fr) and IFNB1 treatment of primary rhesus astrocytes resulted in significant upregulation of SAMHD1 by 48 hours post infection and at all subsequent time points examined. SIV infection alone stimulated apparently significant upregulation at two and 14 days post infection. ***p

    Article Snippet: Pre-designed TaqMan Assays (Applied Biosystems/Life Technologies) were used to quantitate SAMHD1, Mx1, GAPDH, and ACTB from human (Hs00210019_m1, Hs00895608_m1, Hs02758991_g1, and Hs99999903_m1, respectively) and macaque (Rh02869978_m1, Rh02842279_m1, Rh02621745_g1, and Rh03043379_gH, respectively).

    Techniques: Infection, Real-time Polymerase Chain Reaction