plasmid dna  (Promega)

 
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    Name:
    Transfection Carrier DNA
    Description:
    A plasmid DNA used to reduce the amount of an expression vector or reporter vector in mammalian cell transfection while maintaining the overall amount of DNA
    Catalog Number:
    e4881
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    Instrumentation Labware Biochemicals Labware Nucleic Acids
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    Structured Review

    Promega plasmid dna
    Production of monoclonal ORF2p antibodies. a Expression constructs used to generate antigens for ORF2p antibody production. b Coomassie-stained protein electrophoresis gels illustrating purity of ORF2p antigens used in antibody generation. c Immunization strategy to produce rabbit monoclonal antibodies. d Western blot detection of overexpressed ORF2p-3xFlag obtained from HEK-293T LD cells <t>transfected</t> with pLD561 (shown in panel a) using 5 different monoclonal antibodies (Ab) compared to anti-Flag. e Immunoprecipitation of ORF2p-3xFlag using 3 antibodies. f Immunofluorescence imaging of HEK-293T LD cells expressing ORF2p-3xFlag showing co-localization with anti-Flag antibody. g Immunohistochemistry of HEK-293T LD cells expressing ORF2p-3xFlag with 4 monoclonal antibodies compared to anti-Flag. h Above, overview of PhIP-Seq. A phage library expresses protein epitopes from the protein-coding genome, which are affinity purified with ORF2p antibodies. <t>DNA</t> sequences are then isolated and sequenced to identify the genes encoding the peptides. Below, results from five monoclonal antibodies targeting ORF2p. In each instance, the greatest affinity of the ORF2p monoclonal antibodies is for peptides encoded by L1Hs ORF2p peptides. EN = endonuclease, RT = reverse transcriptase, MBP = mannose binding protein, SUMO = small ubiquitin-like modification
    A plasmid DNA used to reduce the amount of an expression vector or reporter vector in mammalian cell transfection while maintaining the overall amount of DNA
    https://www.bioz.com/result/plasmid dna/product/Promega
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid dna - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "LINE-1 ORF2p expression is nearly imperceptible in human cancers"

    Article Title: LINE-1 ORF2p expression is nearly imperceptible in human cancers

    Journal: Mobile DNA

    doi: 10.1186/s13100-019-0191-2

    Production of monoclonal ORF2p antibodies. a Expression constructs used to generate antigens for ORF2p antibody production. b Coomassie-stained protein electrophoresis gels illustrating purity of ORF2p antigens used in antibody generation. c Immunization strategy to produce rabbit monoclonal antibodies. d Western blot detection of overexpressed ORF2p-3xFlag obtained from HEK-293T LD cells transfected with pLD561 (shown in panel a) using 5 different monoclonal antibodies (Ab) compared to anti-Flag. e Immunoprecipitation of ORF2p-3xFlag using 3 antibodies. f Immunofluorescence imaging of HEK-293T LD cells expressing ORF2p-3xFlag showing co-localization with anti-Flag antibody. g Immunohistochemistry of HEK-293T LD cells expressing ORF2p-3xFlag with 4 monoclonal antibodies compared to anti-Flag. h Above, overview of PhIP-Seq. A phage library expresses protein epitopes from the protein-coding genome, which are affinity purified with ORF2p antibodies. DNA sequences are then isolated and sequenced to identify the genes encoding the peptides. Below, results from five monoclonal antibodies targeting ORF2p. In each instance, the greatest affinity of the ORF2p monoclonal antibodies is for peptides encoded by L1Hs ORF2p peptides. EN = endonuclease, RT = reverse transcriptase, MBP = mannose binding protein, SUMO = small ubiquitin-like modification
    Figure Legend Snippet: Production of monoclonal ORF2p antibodies. a Expression constructs used to generate antigens for ORF2p antibody production. b Coomassie-stained protein electrophoresis gels illustrating purity of ORF2p antigens used in antibody generation. c Immunization strategy to produce rabbit monoclonal antibodies. d Western blot detection of overexpressed ORF2p-3xFlag obtained from HEK-293T LD cells transfected with pLD561 (shown in panel a) using 5 different monoclonal antibodies (Ab) compared to anti-Flag. e Immunoprecipitation of ORF2p-3xFlag using 3 antibodies. f Immunofluorescence imaging of HEK-293T LD cells expressing ORF2p-3xFlag showing co-localization with anti-Flag antibody. g Immunohistochemistry of HEK-293T LD cells expressing ORF2p-3xFlag with 4 monoclonal antibodies compared to anti-Flag. h Above, overview of PhIP-Seq. A phage library expresses protein epitopes from the protein-coding genome, which are affinity purified with ORF2p antibodies. DNA sequences are then isolated and sequenced to identify the genes encoding the peptides. Below, results from five monoclonal antibodies targeting ORF2p. In each instance, the greatest affinity of the ORF2p monoclonal antibodies is for peptides encoded by L1Hs ORF2p peptides. EN = endonuclease, RT = reverse transcriptase, MBP = mannose binding protein, SUMO = small ubiquitin-like modification

    Techniques Used: Expressing, Construct, Staining, Protein Electrophoresis, Western Blot, Transfection, Immunoprecipitation, Immunofluorescence, Imaging, Immunohistochemistry, Affinity Purification, Isolation, Binding Assay, Modification

    Related Articles

    Transfection:

    Article Title: SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner
    Article Snippet: The promoter regions of these mutated and truncated reporters were sequenced and aligned against the wild-type reporter to confirm effective mutagenesis. .. HEK cells were plated into six-well plates at a density of 2.5 × 105 cells/well, allowed to adhere to the plate overnight, and transfected with 2.0 μg (including 0.25 μg reporter DNA) of total plasmid DNA per well using FuGene 6 per the manufacturer's recommendations (Promega, Madison, WI). .. An enhanced GFP (eGFP) plasmid was used as a transfection control and to balance total DNA between conditions.

    Article Title: Program Specificity for Ptf1a in Pancreas versus Neural Tube Development Correlates with Distinct Collaborating Cofactors and Chromatin Accessibility
    Article Snippet: .. HEK293 or pancreatic 266-6 cells were transfected with plasmid DNA using FuGene 6 (Promega) as described previously ( ). .. Transfection results reflect at least four independent experiments assayed in duplicate. β-Galactosidase (β-gal) levels expressed by reporter genes were normalized according to the levels of Renilla luciferase from the cotransfected pRL-CMV (Promega).

    Article Title: Interpretation of X Chromosome Dose at Sex-lethal Requires Non-E-Box Sites for the Basic Helix-Loop-Helix Proteins SISB and Daughterless
    Article Snippet: Cultivation, transfection, and assay of Schneider L2 cells were performed according to procedures described by Han et al. ( ). .. One microgram of DNA was used per plate and included 0.1 μg of Fluc reporter, 0.05 μg each of sisB and/or da expression constructs, 0.1 μg of simian virus 40 (SV40) or cytomegalovirus (CMV)- Renilla luciferase reporters to control for transfection efficiency (pRL-SV40 and pRL-CMV; Promega), and carrier DNA. .. Luciferase activity was determined using a Dual-Luciferase assay kit (Promega) and a Berthold Lumat LB9501 luminometer.

    Article Title: RNase H2, mutated in Aicardi‐Goutières syndrome, promotes LINE‐1 retrotransposition
    Article Snippet: For assays employing human LINE‐1‐based constructs (plasmid JM101/L1.3 and mutants, JJ101/L1.3 and mutants, and pXY014 and mutants), approximately 2 × 104 cells were plated per well in a 6‐well dish; when employing zebrafish LINEs (plasmid Zfl2‐2mneoI ) and LTR‐retrotransposons (plasmid pCMVMusD‐6neoTNF ), approximately 4 × 104 cells were plated per well in a 6‐well dish; in toxicity assays (plasmids pU6ineo or pcDNA6.1), approximately 1 × 104 cells were plated per well in a 6‐well dish; in some toxicity assays, approximately 4 × 104 cells were plated in 10‐cm plates. .. Eighteen hours after plating, DNA transfections were carried out using FuGene 6 transfection reagent (Promega) and Opti‐MEM (Life Technologies) following the protocol provided by the manufacturer (for a six‐well plate: 3 μl of FuGene and 97 μl of Opti‐MEM and 1 μg of DNA transfected; for a 10‐cm plate: 12 μl of FuGene and 388 μl of Opti‐MEM and 4 μg of DNA transfected). .. Luciferase‐based retrotransposition assays were analysed 96 h post‐transfection using the Dual‐Glo® luciferase assay system (Promega) following the protocol provided by the manufacturer.

    Article Title: Inhibition of Tat activity by the HEXIM1 protein
    Article Snippet: Subconfluent cell cultures were transfected cell cultures were transfected by a liposome method (LipofectAMINE reagent; Life Technologies, Inc.) in 2 cm/dish in multiwells, using 100 ng of reporter DNA and different amounts of activator plasmid DNA as indicated in the text and 20 ng of Renilla luciferase expression plasmid (pRL-CMV, Promega) for normalization of transfections efficiencies. .. Cells were harvested 48 h after DNA transfections, and cellular extracts were assayed for luciferase activity using Dual-Luciferase Reporter assay (Promega) according to the manufacturer's instructions. .. The experimental reporter luciferase activity was normalized to transfection efficiency as measured by the activity deriving from pRL-CMV.

    Plasmid Preparation:

    Article Title: SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner
    Article Snippet: The promoter regions of these mutated and truncated reporters were sequenced and aligned against the wild-type reporter to confirm effective mutagenesis. .. HEK cells were plated into six-well plates at a density of 2.5 × 105 cells/well, allowed to adhere to the plate overnight, and transfected with 2.0 μg (including 0.25 μg reporter DNA) of total plasmid DNA per well using FuGene 6 per the manufacturer's recommendations (Promega, Madison, WI). .. An enhanced GFP (eGFP) plasmid was used as a transfection control and to balance total DNA between conditions.

    Article Title: Program Specificity for Ptf1a in Pancreas versus Neural Tube Development Correlates with Distinct Collaborating Cofactors and Chromatin Accessibility
    Article Snippet: .. HEK293 or pancreatic 266-6 cells were transfected with plasmid DNA using FuGene 6 (Promega) as described previously ( ). .. Transfection results reflect at least four independent experiments assayed in duplicate. β-Galactosidase (β-gal) levels expressed by reporter genes were normalized according to the levels of Renilla luciferase from the cotransfected pRL-CMV (Promega).

    Real-time Polymerase Chain Reaction:

    Article Title: Reducing persistent polyomavirus infection increases functionality of virus-specific memory CD8 T cells
    Article Snippet: Cells and supernatant were harvested at indicated days p.i. and subjected to freeze-thaw cycles, and the subsequent viral lysate was titrated by plaque assay. .. To quantify viral genomes by quantitative PCR (qPCR), total DNA was extracted from organs or cell cultures with a DNA extraction kit (Promega) and viral genome copies were quantified based on a standard curve of known MuPyV genome copy number vs threshold cycle of detection, as previously described ( ). .. Total RNA was isolated from A2 or A2-Flx infected NMuMG cells using an RNA extraction kit (Promega), then digested with DNase I (Invitrogen) to remove contaminating DNA.

    DNA Extraction:

    Article Title: Reducing persistent polyomavirus infection increases functionality of virus-specific memory CD8 T cells
    Article Snippet: Cells and supernatant were harvested at indicated days p.i. and subjected to freeze-thaw cycles, and the subsequent viral lysate was titrated by plaque assay. .. To quantify viral genomes by quantitative PCR (qPCR), total DNA was extracted from organs or cell cultures with a DNA extraction kit (Promega) and viral genome copies were quantified based on a standard curve of known MuPyV genome copy number vs threshold cycle of detection, as previously described ( ). .. Total RNA was isolated from A2 or A2-Flx infected NMuMG cells using an RNA extraction kit (Promega), then digested with DNase I (Invitrogen) to remove contaminating DNA.

    Expressing:

    Article Title: Interpretation of X Chromosome Dose at Sex-lethal Requires Non-E-Box Sites for the Basic Helix-Loop-Helix Proteins SISB and Daughterless
    Article Snippet: Cultivation, transfection, and assay of Schneider L2 cells were performed according to procedures described by Han et al. ( ). .. One microgram of DNA was used per plate and included 0.1 μg of Fluc reporter, 0.05 μg each of sisB and/or da expression constructs, 0.1 μg of simian virus 40 (SV40) or cytomegalovirus (CMV)- Renilla luciferase reporters to control for transfection efficiency (pRL-SV40 and pRL-CMV; Promega), and carrier DNA. .. Luciferase activity was determined using a Dual-Luciferase assay kit (Promega) and a Berthold Lumat LB9501 luminometer.

    Construct:

    Article Title: Interpretation of X Chromosome Dose at Sex-lethal Requires Non-E-Box Sites for the Basic Helix-Loop-Helix Proteins SISB and Daughterless
    Article Snippet: Cultivation, transfection, and assay of Schneider L2 cells were performed according to procedures described by Han et al. ( ). .. One microgram of DNA was used per plate and included 0.1 μg of Fluc reporter, 0.05 μg each of sisB and/or da expression constructs, 0.1 μg of simian virus 40 (SV40) or cytomegalovirus (CMV)- Renilla luciferase reporters to control for transfection efficiency (pRL-SV40 and pRL-CMV; Promega), and carrier DNA. .. Luciferase activity was determined using a Dual-Luciferase assay kit (Promega) and a Berthold Lumat LB9501 luminometer.

    Luciferase:

    Article Title: Interpretation of X Chromosome Dose at Sex-lethal Requires Non-E-Box Sites for the Basic Helix-Loop-Helix Proteins SISB and Daughterless
    Article Snippet: Cultivation, transfection, and assay of Schneider L2 cells were performed according to procedures described by Han et al. ( ). .. One microgram of DNA was used per plate and included 0.1 μg of Fluc reporter, 0.05 μg each of sisB and/or da expression constructs, 0.1 μg of simian virus 40 (SV40) or cytomegalovirus (CMV)- Renilla luciferase reporters to control for transfection efficiency (pRL-SV40 and pRL-CMV; Promega), and carrier DNA. .. Luciferase activity was determined using a Dual-Luciferase assay kit (Promega) and a Berthold Lumat LB9501 luminometer.

    Article Title: Inhibition of Tat activity by the HEXIM1 protein
    Article Snippet: Subconfluent cell cultures were transfected cell cultures were transfected by a liposome method (LipofectAMINE reagent; Life Technologies, Inc.) in 2 cm/dish in multiwells, using 100 ng of reporter DNA and different amounts of activator plasmid DNA as indicated in the text and 20 ng of Renilla luciferase expression plasmid (pRL-CMV, Promega) for normalization of transfections efficiencies. .. Cells were harvested 48 h after DNA transfections, and cellular extracts were assayed for luciferase activity using Dual-Luciferase Reporter assay (Promega) according to the manufacturer's instructions. .. The experimental reporter luciferase activity was normalized to transfection efficiency as measured by the activity deriving from pRL-CMV.

    Polymerase Chain Reaction:

    Article Title: Organization of the Plasmid cpe Locus in Clostridium perfringens Type A Isolates
    Article Snippet: Primers complementary to sequences present in cpe (CPE-4.5F, 5′-CAGTCCTTAGGTGATGGA-3′) or the IS 1470 -like sequences of F4969 (IS1470-likeR) were used to investigate the DNA region downstream of the plasmid or chromosomal cpe gene in C. perfringens type A isolates. .. This PCR utilized total DNA extracted from specified type A isolates as the template and also included each primer (1 μM final concentration), 0.2 mM deoxynucleotide triphosphates (Promega), and 1 μl of Advantage 2 Taq polymerase (Clontech). .. The reaction mixtures, in a total volume of 50 μl, were placed in a thermal cycler (Techne) and amplified under the conditions described above for the dcm - cpe PCR.

    Article Title: Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus"
    Article Snippet: L. asiaticus’, PCR amplification was carried out by using 16S rDNA-based primer sets OI1/OI2c , in 25 μl reaction volume. .. Each reaction contained 1 μl total DNA, 1X PCR buffer, 1.5 mM MgCl2 , 0.2 mM of dNTP each, 0.2 μM of each primer, 1.25U of GoTaq DNA polymerase (Promega) and nuclease-free water. .. PCR amplification was carried out in My Cycler (Bio-Rad, Hercules, CA, USA) with one cycle of 3 min at 94°C followed by 35 cycles of 30 s at 94°C, 1 min at 58°C, 1.30 min at 72°C, and final extension at 72°C for 10 min.

    Concentration Assay:

    Article Title: Organization of the Plasmid cpe Locus in Clostridium perfringens Type A Isolates
    Article Snippet: Primers complementary to sequences present in cpe (CPE-4.5F, 5′-CAGTCCTTAGGTGATGGA-3′) or the IS 1470 -like sequences of F4969 (IS1470-likeR) were used to investigate the DNA region downstream of the plasmid or chromosomal cpe gene in C. perfringens type A isolates. .. This PCR utilized total DNA extracted from specified type A isolates as the template and also included each primer (1 μM final concentration), 0.2 mM deoxynucleotide triphosphates (Promega), and 1 μl of Advantage 2 Taq polymerase (Clontech). .. The reaction mixtures, in a total volume of 50 μl, were placed in a thermal cycler (Techne) and amplified under the conditions described above for the dcm - cpe PCR.

    Activity Assay:

    Article Title: Inhibition of Tat activity by the HEXIM1 protein
    Article Snippet: Subconfluent cell cultures were transfected cell cultures were transfected by a liposome method (LipofectAMINE reagent; Life Technologies, Inc.) in 2 cm/dish in multiwells, using 100 ng of reporter DNA and different amounts of activator plasmid DNA as indicated in the text and 20 ng of Renilla luciferase expression plasmid (pRL-CMV, Promega) for normalization of transfections efficiencies. .. Cells were harvested 48 h after DNA transfections, and cellular extracts were assayed for luciferase activity using Dual-Luciferase Reporter assay (Promega) according to the manufacturer's instructions. .. The experimental reporter luciferase activity was normalized to transfection efficiency as measured by the activity deriving from pRL-CMV.

    Reporter Assay:

    Article Title: Inhibition of Tat activity by the HEXIM1 protein
    Article Snippet: Subconfluent cell cultures were transfected cell cultures were transfected by a liposome method (LipofectAMINE reagent; Life Technologies, Inc.) in 2 cm/dish in multiwells, using 100 ng of reporter DNA and different amounts of activator plasmid DNA as indicated in the text and 20 ng of Renilla luciferase expression plasmid (pRL-CMV, Promega) for normalization of transfections efficiencies. .. Cells were harvested 48 h after DNA transfections, and cellular extracts were assayed for luciferase activity using Dual-Luciferase Reporter assay (Promega) according to the manufacturer's instructions. .. The experimental reporter luciferase activity was normalized to transfection efficiency as measured by the activity deriving from pRL-CMV.

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    • pgem t easy plasmid dna  (Promega)
    97
    Promega pgem t easy plasmid dna
    Biological activity of recombinant MgPDI2. ( A ) Dithiothreitol (DTT)-mediated insulin reduction by recombinant MgPDI2. ( B ) Potential of recombinant MgPDI2 to protect super-coiled <t>DNA</t> from cleavage in a mixed-function oxidase (MFO) system. Lanes: a, <t>pGEM-T</t> without any treatment; b, pGEM-T incubated with 1.65 mM DTT; c, pGEM-T incubated with 16.5 mM FeCl3; d, pGEM-T incubated with the MFO system; e–h, pGEM-T incubated with the MFO system and different concentrations (0.1, 1, 10, and 100 μg/mL, respectively) of purified MgPDI2. The nicked, linear, and super-coiled forms of pGEM-T are shown from the top to the bottom. Numerical values are reported in Table 1 .
    Pgem T Easy Plasmid Dna, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgem t easy plasmid dna/product/Promega
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pgem t easy plasmid dna - by Bioz Stars, 2021-03
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    plasmid dna purification system  (Promega)
    97
    Promega plasmid dna purification system
    Hepatitis B viral virus X protein (HBx) X1 and X2 fragments amplified by PCR from <t>AH109-pAS2-1-X1</t> and AH109-pAS2-1-X2. X1 and X2 fragments of the expected size can be seen after gel electrophoresis (0.6% agarose). Lane 1, 100-bp <t>DNA</t> ladder; lanes 2–4, HBx X1 (216 bp) amplified with primers P1 and P2; lane 5, negative controls; lanes 6 and 7, HBx X2 (352 bp) amplified with primers P1 and P3.
    Plasmid Dna Purification System, supplied by Promega, 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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    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid dna purification system - by Bioz Stars, 2021-03
    97/100 stars
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    firefly luciferase reporter plasmid dna  (Promega)
    99
    Promega firefly luciferase reporter plasmid dna
    Jade1 is transcriptionally induced after <t>DNA</t> damage. ( A ) Jade1protein is induced after DNA damage. ( B ) Jade1 expression is slightly reduced in Atm−/− MEFs. ( C ) The level of Jade1mRNA is induced in an ATM-dependent manner after DNA damage. P21 mRNA is served as an indicator for the functionality of the DNA damage response. ( D ) Activity of the Jade1 promoter is upregulated in ATM-dependent manner after DNA damage. MCF-7 cells were <t>transfected</t> with the Jade1 promoter-driven firefly luciferase expression vector and Renilla luciferase expression vector. Firefly luciferase activity was measured and normalized to the activity of Renilla luciferase. ( E ) Knockdown of ATM in MCF7 cells inhibits Jade1 expression. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d. page.
    Firefly Luciferase Reporter Plasmid Dna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/firefly luciferase reporter plasmid dna/product/Promega
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    firefly luciferase reporter plasmid dna - by Bioz Stars, 2021-03
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    luciferase reporter plasmid dna  (Promega)
    99
    Promega luciferase reporter plasmid dna
    Impact of protein expression compared with the loss of N terminus on mutant protein function. ( a ) HEK293T cells were transfected with a set amount (0.5 μ g) of plasmid <t>DNA,</t> and 24 h <t>posttransfection</t> the resulting protein lysates
    Luciferase Reporter Plasmid Dna, supplied by Promega, 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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    Biological activity of recombinant MgPDI2. ( A ) Dithiothreitol (DTT)-mediated insulin reduction by recombinant MgPDI2. ( B ) Potential of recombinant MgPDI2 to protect super-coiled DNA from cleavage in a mixed-function oxidase (MFO) system. Lanes: a, pGEM-T without any treatment; b, pGEM-T incubated with 1.65 mM DTT; c, pGEM-T incubated with 16.5 mM FeCl3; d, pGEM-T incubated with the MFO system; e–h, pGEM-T incubated with the MFO system and different concentrations (0.1, 1, 10, and 100 μg/mL, respectively) of purified MgPDI2. The nicked, linear, and super-coiled forms of pGEM-T are shown from the top to the bottom. Numerical values are reported in Table 1 .

    Journal: International Journal of Molecular Sciences

    Article Title: Identification and Characterization of a Novel Protein Disulfide Isomerase Gene (MgPDI2) from Meloidogyne graminicola

    doi: 10.3390/ijms21249586

    Figure Lengend Snippet: Biological activity of recombinant MgPDI2. ( A ) Dithiothreitol (DTT)-mediated insulin reduction by recombinant MgPDI2. ( B ) Potential of recombinant MgPDI2 to protect super-coiled DNA from cleavage in a mixed-function oxidase (MFO) system. Lanes: a, pGEM-T without any treatment; b, pGEM-T incubated with 1.65 mM DTT; c, pGEM-T incubated with 16.5 mM FeCl3; d, pGEM-T incubated with the MFO system; e–h, pGEM-T incubated with the MFO system and different concentrations (0.1, 1, 10, and 100 μg/mL, respectively) of purified MgPDI2. The nicked, linear, and super-coiled forms of pGEM-T are shown from the top to the bottom. Numerical values are reported in Table 1 .

    Article Snippet: Briefly, the mixed-function oxidase (MFO) system, consisting of 1.65 mM DTT, 16.5 mM FeCl3, and different concentrations of MgPDI2 (from 0.1 to 100 µg/mL) were pre-incubated at 37 °C for 2.5 h. pGEM-T easy plasmid DNA (500 ng, Promega, Madison, WI, USA) was then added and incubated for 1 h at 37 °C.

    Techniques: Activity Assay, Recombinant, Incubation, Purification

    Hepatitis B viral virus X protein (HBx) X1 and X2 fragments amplified by PCR from AH109-pAS2-1-X1 and AH109-pAS2-1-X2. X1 and X2 fragments of the expected size can be seen after gel electrophoresis (0.6% agarose). Lane 1, 100-bp DNA ladder; lanes 2–4, HBx X1 (216 bp) amplified with primers P1 and P2; lane 5, negative controls; lanes 6 and 7, HBx X2 (352 bp) amplified with primers P1 and P3.

    Journal: International Journal of Molecular Medicine

    Article Title: Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III

    doi: 10.3892/ijmm.2014.2018

    Figure Lengend Snippet: Hepatitis B viral virus X protein (HBx) X1 and X2 fragments amplified by PCR from AH109-pAS2-1-X1 and AH109-pAS2-1-X2. X1 and X2 fragments of the expected size can be seen after gel electrophoresis (0.6% agarose). Lane 1, 100-bp DNA ladder; lanes 2–4, HBx X1 (216 bp) amplified with primers P1 and P2; lane 5, negative controls; lanes 6 and 7, HBx X2 (352 bp) amplified with primers P1 and P3.

    Article Snippet: The pAS2-1 plasmid was extracted from E. coli DH5α cells using the Plasmid DNA purification system, following the manufacturer’s instructions (Promega Biosciences LLC).

    Techniques: Amplification, Polymerase Chain Reaction, Nucleic Acid Electrophoresis

    Hepatitis B viral virus X protein (HBx) fragments amplified by PCR from pAS2-1 recombinant plasmid and separated by gel electrophoresis (0.6% agarose). The different fragments have the expected molecular weights. Lane 1, 100-bp DNA ladder used as molecular weight marker; lane 2, full length (464 bp) HBx gene; lane 3, HBx X2 (352 bp) fragment; lane 4, negative controls; lanes 5–7, HBx X1 (216 bp) fragment.

    Journal: International Journal of Molecular Medicine

    Article Title: Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III

    doi: 10.3892/ijmm.2014.2018

    Figure Lengend Snippet: Hepatitis B viral virus X protein (HBx) fragments amplified by PCR from pAS2-1 recombinant plasmid and separated by gel electrophoresis (0.6% agarose). The different fragments have the expected molecular weights. Lane 1, 100-bp DNA ladder used as molecular weight marker; lane 2, full length (464 bp) HBx gene; lane 3, HBx X2 (352 bp) fragment; lane 4, negative controls; lanes 5–7, HBx X1 (216 bp) fragment.

    Article Snippet: The pAS2-1 plasmid was extracted from E. coli DH5α cells using the Plasmid DNA purification system, following the manufacturer’s instructions (Promega Biosciences LLC).

    Techniques: Amplification, Polymerase Chain Reaction, Recombinant, Plasmid Preparation, Nucleic Acid Electrophoresis, Molecular Weight, Marker

    (A) DNA sequence of hepatitis B viral virus X protein (HBx) X1. Amplified HBx X1 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X1. Purified pAS2-1-X1 was sent to Life Technologies Corp. for sequencing. The X1 gene has 98% homology with the X gene from the GenBank database. The X1 gene runs from base 63 to base 278. (B) DNA sequence of HBx X2. Amplified HBx X2 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X2. The sequence shows that the X2 gene has 98% homology with X gene from the GenBank database. The X2 gene runs from base 61 to base 412.

    Journal: International Journal of Molecular Medicine

    Article Title: Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III

    doi: 10.3892/ijmm.2014.2018

    Figure Lengend Snippet: (A) DNA sequence of hepatitis B viral virus X protein (HBx) X1. Amplified HBx X1 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X1. Purified pAS2-1-X1 was sent to Life Technologies Corp. for sequencing. The X1 gene has 98% homology with the X gene from the GenBank database. The X1 gene runs from base 63 to base 278. (B) DNA sequence of HBx X2. Amplified HBx X2 fragments were ligated with linearized pAS2-1 and the recombinant plasmid designated pAS2-1-X2. The sequence shows that the X2 gene has 98% homology with X gene from the GenBank database. The X2 gene runs from base 61 to base 412.

    Article Snippet: The pAS2-1 plasmid was extracted from E. coli DH5α cells using the Plasmid DNA purification system, following the manufacturer’s instructions (Promega Biosciences LLC).

    Techniques: Sequencing, Amplification, Recombinant, Plasmid Preparation, Purification

    Jade1 is transcriptionally induced after DNA damage. ( A ) Jade1protein is induced after DNA damage. ( B ) Jade1 expression is slightly reduced in Atm−/− MEFs. ( C ) The level of Jade1mRNA is induced in an ATM-dependent manner after DNA damage. P21 mRNA is served as an indicator for the functionality of the DNA damage response. ( D ) Activity of the Jade1 promoter is upregulated in ATM-dependent manner after DNA damage. MCF-7 cells were transfected with the Jade1 promoter-driven firefly luciferase expression vector and Renilla luciferase expression vector. Firefly luciferase activity was measured and normalized to the activity of Renilla luciferase. ( E ) Knockdown of ATM in MCF7 cells inhibits Jade1 expression. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d. page.

    Journal: The EMBO Journal

    Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    doi: 10.1038/emboj.2013.221

    Figure Lengend Snippet: Jade1 is transcriptionally induced after DNA damage. ( A ) Jade1protein is induced after DNA damage. ( B ) Jade1 expression is slightly reduced in Atm−/− MEFs. ( C ) The level of Jade1mRNA is induced in an ATM-dependent manner after DNA damage. P21 mRNA is served as an indicator for the functionality of the DNA damage response. ( D ) Activity of the Jade1 promoter is upregulated in ATM-dependent manner after DNA damage. MCF-7 cells were transfected with the Jade1 promoter-driven firefly luciferase expression vector and Renilla luciferase expression vector. Firefly luciferase activity was measured and normalized to the activity of Renilla luciferase. ( E ) Knockdown of ATM in MCF7 cells inhibits Jade1 expression. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d. page.

    Article Snippet: Human U2OS cells were transfected with 200 ng of firefly luciferase reporter plasmid DNA (Control, lncRNA-JADE or Jade1 promoter-driven luciferase construct) and 0.6 ng of Renilla luciferase reporter plasmid pRL-TK (Promega) per well in 24-well dish.

    Techniques: Expressing, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Polyacrylamide Gel Electrophoresis

    LncRNA-JADE is induced after DNA damage. ( A ) Schematic illustration showing Jade1 and lncRNA-JADE genes in mouse and human. ( B ) Mouse and human lncRNA-JADE are induced in an ATM-dependent manner after DNA damage. ( C ) DNA damage positively regulates lncRNA-JADE promoter activity in an ATM-dependent manner. ATM-IN: ATM inhibitor. ( D ) Schematic illustration showing the NF-κB binding elements in the Jade1 promoter. DNA damage induces the activity of lncRNA-JADE promoter in an NF-κB-dependent manner. NF-κB-IN: NF-κB inhibitor. ( E ) Expression of LncRNA-JADE is regulated by ATM and NF-κB after DNA damage. IRF-1 (interferon response factor-1) is served as a positive control in the NF-κB signalling. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d.

    Journal: The EMBO Journal

    Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    doi: 10.1038/emboj.2013.221

    Figure Lengend Snippet: LncRNA-JADE is induced after DNA damage. ( A ) Schematic illustration showing Jade1 and lncRNA-JADE genes in mouse and human. ( B ) Mouse and human lncRNA-JADE are induced in an ATM-dependent manner after DNA damage. ( C ) DNA damage positively regulates lncRNA-JADE promoter activity in an ATM-dependent manner. ATM-IN: ATM inhibitor. ( D ) Schematic illustration showing the NF-κB binding elements in the Jade1 promoter. DNA damage induces the activity of lncRNA-JADE promoter in an NF-κB-dependent manner. NF-κB-IN: NF-κB inhibitor. ( E ) Expression of LncRNA-JADE is regulated by ATM and NF-κB after DNA damage. IRF-1 (interferon response factor-1) is served as a positive control in the NF-κB signalling. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d.

    Article Snippet: Human U2OS cells were transfected with 200 ng of firefly luciferase reporter plasmid DNA (Control, lncRNA-JADE or Jade1 promoter-driven luciferase construct) and 0.6 ng of Renilla luciferase reporter plasmid pRL-TK (Promega) per well in 24-well dish.

    Techniques: Activity Assay, Binding Assay, Expressing, Positive Control

    Brca1 binds lncRNA-JADE and mediates Jade1 induction in the DNA damage response via p300-containing transcription complex. ( A ) Schematic illustration showing the p300/CBP binding elements in the Jade1 promoter. ( B ) p300 physically interacts with the promoter region of Jade1 gene. Control or lncRNA-JADE knockdown MCF7 cells were treated with or without NCS (200 ng/ml) and cell lysates were immunoprecipitated with control IgG or p300 antibodies. The p300-binding activity of Jade1 promoter DNA was quantified by qPCR. ( C ) Brca1 interacts with p300 and this interaction is increased after DNA damage. ( D ) Jade1 promoter activity is induced in a Brca1-dependent manner after DNA damage. MCF-7 cells were infected with lentiviruses expressing control or Brca1 shRNA. The cells were transfected with pGL3-control vector (SV40 promoter) or Jade1 promoter-driven firefly luciferase expression vector and Renilla luciferase expression vector 2 days post infection. They were treated with NCS (500 ng/ml) 24 h after transfection and then harvested 16 h after treatment. Firefly luciferase activity was measured and normalized to the activity of Renilla luciferase. ( E ) LncRNA-JADE physically interacts with Brca1. 5′- and 3′-deletion mutants of lncRNA-JADE were generated as indicated. Two pairs of primers were used to detect the Brca1-binding sequences of lncRNA-JADE in RIP assays. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d. page.

    Journal: The EMBO Journal

    Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    doi: 10.1038/emboj.2013.221

    Figure Lengend Snippet: Brca1 binds lncRNA-JADE and mediates Jade1 induction in the DNA damage response via p300-containing transcription complex. ( A ) Schematic illustration showing the p300/CBP binding elements in the Jade1 promoter. ( B ) p300 physically interacts with the promoter region of Jade1 gene. Control or lncRNA-JADE knockdown MCF7 cells were treated with or without NCS (200 ng/ml) and cell lysates were immunoprecipitated with control IgG or p300 antibodies. The p300-binding activity of Jade1 promoter DNA was quantified by qPCR. ( C ) Brca1 interacts with p300 and this interaction is increased after DNA damage. ( D ) Jade1 promoter activity is induced in a Brca1-dependent manner after DNA damage. MCF-7 cells were infected with lentiviruses expressing control or Brca1 shRNA. The cells were transfected with pGL3-control vector (SV40 promoter) or Jade1 promoter-driven firefly luciferase expression vector and Renilla luciferase expression vector 2 days post infection. They were treated with NCS (500 ng/ml) 24 h after transfection and then harvested 16 h after treatment. Firefly luciferase activity was measured and normalized to the activity of Renilla luciferase. ( E ) LncRNA-JADE physically interacts with Brca1. 5′- and 3′-deletion mutants of lncRNA-JADE were generated as indicated. Two pairs of primers were used to detect the Brca1-binding sequences of lncRNA-JADE in RIP assays. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d. page.

    Article Snippet: Human U2OS cells were transfected with 200 ng of firefly luciferase reporter plasmid DNA (Control, lncRNA-JADE or Jade1 promoter-driven luciferase construct) and 0.6 ng of Renilla luciferase reporter plasmid pRL-TK (Promega) per well in 24-well dish.

    Techniques: Binding Assay, Immunoprecipitation, Activity Assay, Real-time Polymerase Chain Reaction, Infection, Expressing, shRNA, Transfection, Plasmid Preparation, Luciferase, Generated, Polyacrylamide Gel Electrophoresis

    LncRNA-JADE positively regulates histone H4 acetylation through Jade1. ( A ) H4 acetylation is induced after DNA damage. MCF7 cells were treated with NCS (500 ng/ml). H4Ac: total histone H4 acetylation; K5, K8, K12: Histone H4 acetylation at lysine 5, 8, or 12. ( B ) Jade1 knockdown abolishes the induction of H4 acetylation after DNA damage. ( C ) LncRNA-JADE positively regulates H4 acetylation and Jade1. Overexpression of lncRNA-JADE enhanced the induction of H4 acetylation and Jade1, and knockdown of lncRNA-JADE abolished the induction of H4 acetylation and Jade1. Semi-quantification of proteins is shown at the bottom. page.

    Journal: The EMBO Journal

    Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    doi: 10.1038/emboj.2013.221

    Figure Lengend Snippet: LncRNA-JADE positively regulates histone H4 acetylation through Jade1. ( A ) H4 acetylation is induced after DNA damage. MCF7 cells were treated with NCS (500 ng/ml). H4Ac: total histone H4 acetylation; K5, K8, K12: Histone H4 acetylation at lysine 5, 8, or 12. ( B ) Jade1 knockdown abolishes the induction of H4 acetylation after DNA damage. ( C ) LncRNA-JADE positively regulates H4 acetylation and Jade1. Overexpression of lncRNA-JADE enhanced the induction of H4 acetylation and Jade1, and knockdown of lncRNA-JADE abolished the induction of H4 acetylation and Jade1. Semi-quantification of proteins is shown at the bottom. page.

    Article Snippet: Human U2OS cells were transfected with 200 ng of firefly luciferase reporter plasmid DNA (Control, lncRNA-JADE or Jade1 promoter-driven luciferase construct) and 0.6 ng of Renilla luciferase reporter plasmid pRL-TK (Promega) per well in 24-well dish.

    Techniques: Over Expression, Polyacrylamide Gel Electrophoresis

    Biological functions of lncRNA-JADE in human MCF7 cells. ( A ) LncRNA-JADE positively regulates MCF7 cell proliferation. ( B ) Altering lncRNA-JADE expression affects DNA damage-induced cell-cycle arrest. Cell-cycle profiles were analysed by flow cytometry using propidium iodide-stained cells. ( C ) Knockdown of lncRNA-JADE increases cell apoptosis in the control and NCS-treated cells. The percentage of TUNEL-positive cells was summarized in the graph. ( D ) Knockdown of lncRNA-JADE increases the cell sensitivity to DNA damaging drugs NCS, Etopside, and Bleomycin. MCF7 cells were treated with DNA damaging agents as indicated and cultured for 48 h and cell viability was measured. Graphic data in this figure present the mean of three biological replicates and error bars depict s.d.

    Journal: The EMBO Journal

    Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    doi: 10.1038/emboj.2013.221

    Figure Lengend Snippet: Biological functions of lncRNA-JADE in human MCF7 cells. ( A ) LncRNA-JADE positively regulates MCF7 cell proliferation. ( B ) Altering lncRNA-JADE expression affects DNA damage-induced cell-cycle arrest. Cell-cycle profiles were analysed by flow cytometry using propidium iodide-stained cells. ( C ) Knockdown of lncRNA-JADE increases cell apoptosis in the control and NCS-treated cells. The percentage of TUNEL-positive cells was summarized in the graph. ( D ) Knockdown of lncRNA-JADE increases the cell sensitivity to DNA damaging drugs NCS, Etopside, and Bleomycin. MCF7 cells were treated with DNA damaging agents as indicated and cultured for 48 h and cell viability was measured. Graphic data in this figure present the mean of three biological replicates and error bars depict s.d.

    Article Snippet: Human U2OS cells were transfected with 200 ng of firefly luciferase reporter plasmid DNA (Control, lncRNA-JADE or Jade1 promoter-driven luciferase construct) and 0.6 ng of Renilla luciferase reporter plasmid pRL-TK (Promega) per well in 24-well dish.

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, TUNEL Assay, Cell Culture

    ATM-dependent regulation of lncRNA expression in response to DNA damage. ( A ) Experimental layout to identify ATM-dependent lncRNAs. Atm+/+ and Atm−/− mouse embryonic fibroblasts (MEFs) were treated with NCS (200 ng/ml) and harvested at indicated time points for microarray analyses. ( B ) The number of ATM-dependent lncRNAs upon DNA damage. ( C ) A representative group of ATM-dependent and DNA damage-induced lncRNAs. Green or red colour on the heat map indicates a decrease or an increase in the lncRNA level and colour intensities correspond to relative signal levels on a logarithmic scale. ( D ) Quantitative PCR validation of representative lncRNAs. Data represent the mean of three experimental replicates, with error bars depicting s.d.

    Journal: The EMBO Journal

    Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    doi: 10.1038/emboj.2013.221

    Figure Lengend Snippet: ATM-dependent regulation of lncRNA expression in response to DNA damage. ( A ) Experimental layout to identify ATM-dependent lncRNAs. Atm+/+ and Atm−/− mouse embryonic fibroblasts (MEFs) were treated with NCS (200 ng/ml) and harvested at indicated time points for microarray analyses. ( B ) The number of ATM-dependent lncRNAs upon DNA damage. ( C ) A representative group of ATM-dependent and DNA damage-induced lncRNAs. Green or red colour on the heat map indicates a decrease or an increase in the lncRNA level and colour intensities correspond to relative signal levels on a logarithmic scale. ( D ) Quantitative PCR validation of representative lncRNAs. Data represent the mean of three experimental replicates, with error bars depicting s.d.

    Article Snippet: Human U2OS cells were transfected with 200 ng of firefly luciferase reporter plasmid DNA (Control, lncRNA-JADE or Jade1 promoter-driven luciferase construct) and 0.6 ng of Renilla luciferase reporter plasmid pRL-TK (Promega) per well in 24-well dish.

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

    Impact of protein expression compared with the loss of N terminus on mutant protein function. ( a ) HEK293T cells were transfected with a set amount (0.5 μ g) of plasmid DNA, and 24 h posttransfection the resulting protein lysates

    Journal: European Journal of Human Genetics

    Article Title: Reinitiation of mRNA translation in a patient with X-linked infantile spasms with a protein-truncating variant in ARX

    doi: 10.1038/ejhg.2015.176

    Figure Lengend Snippet: Impact of protein expression compared with the loss of N terminus on mutant protein function. ( a ) HEK293T cells were transfected with a set amount (0.5 μ g) of plasmid DNA, and 24 h posttransfection the resulting protein lysates

    Article Snippet: Cells were harvested 24 h posttransfection with 500 ng luciferase reporter plasmid DNA, 10 ng pGL4.74[ hRluc /TK] plasmid DNA (Promega) and variable amounts of Myc expression plasmid DNA containing full-length ARX-Wt or premature truncation mutation sequence using Lipofectamine 2000 (Life-Technologies).

    Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation