pcmv gluc  (New England Biolabs)


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    Name:
    pCMV GLuc Control Plasmid
    Description:
    pCMV GLuc Control Plasmid 20 ug
    Catalog Number:
    N8081S
    Price:
    313
    Category:
    Cellular Biology
    Size:
    20 ug
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    Structured Review

    New England Biolabs pcmv gluc
    pCMV GLuc Control Plasmid
    pCMV GLuc Control Plasmid 20 ug
    https://www.bioz.com/result/pcmv gluc/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcmv gluc - by Bioz Stars, 2021-06
    98/100 stars

    Images

    1) Product Images from "Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase"

    Article Title: Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

    Journal: Journal of Virology

    doi: 10.1128/JVI.05871-11

    Effect of brefeldin A on GLuc secretion. (A) Schematic representation of the GLuc-expressing reporter construct pCMV-GLuc. (B) Inhibition of GLuc transport in the presence of the indicated concentrations of BFA. HeLa cells were transfected with a GLuc-expressing vector. At 6 h posttransfection, cells were treated with different amounts of BFA. Samples taken from growth medium and lysates were assayed for GLuc activity at 16 h after addition of BFA.
    Figure Legend Snippet: Effect of brefeldin A on GLuc secretion. (A) Schematic representation of the GLuc-expressing reporter construct pCMV-GLuc. (B) Inhibition of GLuc transport in the presence of the indicated concentrations of BFA. HeLa cells were transfected with a GLuc-expressing vector. At 6 h posttransfection, cells were treated with different amounts of BFA. Samples taken from growth medium and lysates were assayed for GLuc activity at 16 h after addition of BFA.

    Techniques Used: Expressing, Construct, Inhibition, Transfection, Plasmid Preparation, Activity Assay

    2) Product Images from "Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein"

    Article Title: Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01459-18

    Functional impact of GBF1-NS3 interaction on secretion and NS3 protease activity. (A) HeLa cells were cotransfected with pCMV-GLuc and increasing amounts of pcDNA3.1-HA-NS3-4A as indicated. The total amount of DNA was kept constant by adding empty pcDNA3.1. At 16 h posttransfection, the culture medium was changed and the cells were further incubated for 4 h. As a control, cells were treated with 1 μg/ml BFA during this 4-h secretion period. Luciferase activity was measured in supernatants and cell lysates. (B and C) HeLa cells were cotransfected with pEGFP-IPS, pcDNA3.1 HA-NS3-4A, and increasing concentrations of pEYFP-GBF1 as indicated. Empty pcDNA3.1 plasmid was used to keep the same final concentration of total transfected DNA constant. At 24 h posttransfection, EGFP-IPS cleavage was monitored by immunoblotting using anti-GFP antibody (B), and the intensities of bands corresponding to cleaved and uncleaved GPF-IPS were measured and the percentage of cleavage was calculated (C). Error bars represent the standard deviations for 3 independent experiments. *, **, and *** correspond to P values below 0.05, 0.01, and 0.001, respectively.
    Figure Legend Snippet: Functional impact of GBF1-NS3 interaction on secretion and NS3 protease activity. (A) HeLa cells were cotransfected with pCMV-GLuc and increasing amounts of pcDNA3.1-HA-NS3-4A as indicated. The total amount of DNA was kept constant by adding empty pcDNA3.1. At 16 h posttransfection, the culture medium was changed and the cells were further incubated for 4 h. As a control, cells were treated with 1 μg/ml BFA during this 4-h secretion period. Luciferase activity was measured in supernatants and cell lysates. (B and C) HeLa cells were cotransfected with pEGFP-IPS, pcDNA3.1 HA-NS3-4A, and increasing concentrations of pEYFP-GBF1 as indicated. Empty pcDNA3.1 plasmid was used to keep the same final concentration of total transfected DNA constant. At 24 h posttransfection, EGFP-IPS cleavage was monitored by immunoblotting using anti-GFP antibody (B), and the intensities of bands corresponding to cleaved and uncleaved GPF-IPS were measured and the percentage of cleavage was calculated (C). Error bars represent the standard deviations for 3 independent experiments. *, **, and *** correspond to P values below 0.05, 0.01, and 0.001, respectively.

    Techniques Used: Functional Assay, Activity Assay, Incubation, Luciferase, Plasmid Preparation, Concentration Assay, Transfection

    3) Product Images from "In Vitro Screening for Compounds That Enhance Human L1 Mobilization"

    Article Title: In Vitro Screening for Compounds That Enhance Human L1 Mobilization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074629

    A novel secreted Gaussia luciferase L1 retrotransposition assay system. (A) A novel Gaussia luciferase cassette in a human L1. The mGLucI retrotransposition cassette was cloned into the 3′ UTR of L1 RP in the antisense orientation. The cassette contains the GLuc gene interrupted by an antisense mini-intron with an EF-1α/eIF4g hybrid promoter (phEF1/peIF4g) and the TK poly(A) signal (TK pA). GLuc can be expressed only when retrotransposition occurs. (B) Coexpression in HEK293T cells of selected cDNA constructs, previously determined to alter retrotransposition levels in the EGFP reporter assay (right panel; [40] ), similarly affect retrotransposition in the GLuc assay (left panel). Fifty µl of media were sampled from each well (four replicate wells for each construct) at the indicated hourly time points over the course of 10 days and tested at the conclusion of the experiment (RLU: Relative Light Units). EGFP assays were performed at approximately 115 hours and results were normalized to cotransfected 99-PUR-RPS-EGFP reporter construct and empty vector (pcDNA3). Data for the earliest time-points sampled are reproduced in Fig. S3 . (C) Retrotransposition in HeLa cells is confirmed by PCR using primers that flank the intron of the GLuc reporter gene. The presence of a band of 149 bp is diagnostic for intron removal. A 283-bp band is amplified from the transfected 99-PUR-RPS- mGLucI or 99-PUR-JM111- mGLucI plasmid. (D) Immunofluorescence analysis of transfected and fixed HEK293T cells showing obvious expression of GLuc from retrotransposition events formed by 99-PUR-RPS- mGLucI but not 99-PUR-JM111 -mGLucI . Constitutive luciferase expression from pCMV-GLuc is also shown (right), detected by α-Gaussia antibody.
    Figure Legend Snippet: A novel secreted Gaussia luciferase L1 retrotransposition assay system. (A) A novel Gaussia luciferase cassette in a human L1. The mGLucI retrotransposition cassette was cloned into the 3′ UTR of L1 RP in the antisense orientation. The cassette contains the GLuc gene interrupted by an antisense mini-intron with an EF-1α/eIF4g hybrid promoter (phEF1/peIF4g) and the TK poly(A) signal (TK pA). GLuc can be expressed only when retrotransposition occurs. (B) Coexpression in HEK293T cells of selected cDNA constructs, previously determined to alter retrotransposition levels in the EGFP reporter assay (right panel; [40] ), similarly affect retrotransposition in the GLuc assay (left panel). Fifty µl of media were sampled from each well (four replicate wells for each construct) at the indicated hourly time points over the course of 10 days and tested at the conclusion of the experiment (RLU: Relative Light Units). EGFP assays were performed at approximately 115 hours and results were normalized to cotransfected 99-PUR-RPS-EGFP reporter construct and empty vector (pcDNA3). Data for the earliest time-points sampled are reproduced in Fig. S3 . (C) Retrotransposition in HeLa cells is confirmed by PCR using primers that flank the intron of the GLuc reporter gene. The presence of a band of 149 bp is diagnostic for intron removal. A 283-bp band is amplified from the transfected 99-PUR-RPS- mGLucI or 99-PUR-JM111- mGLucI plasmid. (D) Immunofluorescence analysis of transfected and fixed HEK293T cells showing obvious expression of GLuc from retrotransposition events formed by 99-PUR-RPS- mGLucI but not 99-PUR-JM111 -mGLucI . Constitutive luciferase expression from pCMV-GLuc is also shown (right), detected by α-Gaussia antibody.

    Techniques Used: Luciferase, Clone Assay, Construct, Reporter Assay, Plasmid Preparation, Polymerase Chain Reaction, Diagnostic Assay, Amplification, Transfection, Immunofluorescence, Expressing

    4) Product Images from "Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein"

    Article Title: Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01459-18

    Functional impact of GBF1-NS3 interaction on secretion and NS3 protease activity. (A) HeLa cells were cotransfected with pCMV-GLuc and increasing amounts of pcDNA3.1-HA-NS3-4A as indicated. The total amount of DNA was kept constant by adding empty pcDNA3.1. At 16 h posttransfection, the culture medium was changed and the cells were further incubated for 4 h. As a control, cells were treated with 1 μg/ml BFA during this 4-h secretion period. Luciferase activity was measured in supernatants and cell lysates. (B and C) HeLa cells were cotransfected with pEGFP-IPS, pcDNA3.1 HA-NS3-4A, and increasing concentrations of pEYFP-GBF1 as indicated. Empty pcDNA3.1 plasmid was used to keep the same final concentration of total transfected DNA constant. At 24 h posttransfection, EGFP-IPS cleavage was monitored by immunoblotting using anti-GFP antibody (B), and the intensities of bands corresponding to cleaved and uncleaved GPF-IPS were measured and the percentage of cleavage was calculated (C). Error bars represent the standard deviations for 3 independent experiments. *, **, and *** correspond to P values below 0.05, 0.01, and 0.001, respectively.
    Figure Legend Snippet: Functional impact of GBF1-NS3 interaction on secretion and NS3 protease activity. (A) HeLa cells were cotransfected with pCMV-GLuc and increasing amounts of pcDNA3.1-HA-NS3-4A as indicated. The total amount of DNA was kept constant by adding empty pcDNA3.1. At 16 h posttransfection, the culture medium was changed and the cells were further incubated for 4 h. As a control, cells were treated with 1 μg/ml BFA during this 4-h secretion period. Luciferase activity was measured in supernatants and cell lysates. (B and C) HeLa cells were cotransfected with pEGFP-IPS, pcDNA3.1 HA-NS3-4A, and increasing concentrations of pEYFP-GBF1 as indicated. Empty pcDNA3.1 plasmid was used to keep the same final concentration of total transfected DNA constant. At 24 h posttransfection, EGFP-IPS cleavage was monitored by immunoblotting using anti-GFP antibody (B), and the intensities of bands corresponding to cleaved and uncleaved GPF-IPS were measured and the percentage of cleavage was calculated (C). Error bars represent the standard deviations for 3 independent experiments. *, **, and *** correspond to P values below 0.05, 0.01, and 0.001, respectively.

    Techniques Used: Functional Assay, Activity Assay, Incubation, Luciferase, Plasmid Preparation, Concentration Assay, Transfection

    5) Product Images from "Contrasting Roles of Mitogen-Activated Protein Kinases in Cellular Entry and Replication of Hepatitis C Virus: MKNK1 Facilitates Cell Entry"

    Article Title: Contrasting Roles of Mitogen-Activated Protein Kinases in Cellular Entry and Replication of Hepatitis C Virus: MKNK1 Facilitates Cell Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.00954-12

    MKNK inhibition and cellular entry of HCVpp. (A) Impact of increasing concentrations of RO4475417 (MKNK inhibitor) on luciferase expressed by HCVpp versus VSVpp. Cells were treated with the inhibitor for 1 h prior to and for 6 h during pseudotyped particle infection. Luciferase activity was assayed at 48 h, as shown at the top. Results shown represent mean ± SD relative luciferase activity in triplicate cultures and are representative of two independent experiments. (B) Inhibition of HCVpp-mediated luciferase expression by RO4475417 when added to cell culture media 1 h prior to and during HCVpp infection (−1 to 6 h, as described for panel A) or following a 6-h incubation with HCVpp (6 to 12 h). Results shown represent mean ± SE relative luciferase activity. (C) Inhibition of cap-dependent and HCV IRES-dependent translation following a 6-h exposure to increasing concentrations of RO4475417 (MKNK inhibitor). Cells were transfected with pRLHL, which expresses a dicistronic transcript containing the HCV IRES within the intercistronic space. Twenty-four h later (time point 0), cells were treated with RO4475417 for 6 h. Cells were lysed and luciferase activities measured at 48 h. Results shown represent RLuc (cap-dependent translation initiation) and FLuc (IRES-dependent) activities relative to those in the absence of the compound (100%) and are the means ± SD from triplicate cultures. (D) Kinetic analysis of recovery from RO4475417-mediated inhibition of cap-dependent translation. Cells were transfected with pCMV-GLuc, which expresses secreted GLuc, 24 h prior to drug exposure, as described for panel C. Supernatant fluids were harvested for GLuc assay and replaced with fresh medium at 6, 24, and 48 h. (E) Lack of an effect of a 6-h exposure to RO4475417 (5 μM) on cellular abundance of the HCV coreceptors CD81, SR-B1, claudin-1, occludin, and NPC1L1. Cells were treated with compound for 6 h starting at time point 0 and harvested for immunoblot assays at 6 and 24 h.
    Figure Legend Snippet: MKNK inhibition and cellular entry of HCVpp. (A) Impact of increasing concentrations of RO4475417 (MKNK inhibitor) on luciferase expressed by HCVpp versus VSVpp. Cells were treated with the inhibitor for 1 h prior to and for 6 h during pseudotyped particle infection. Luciferase activity was assayed at 48 h, as shown at the top. Results shown represent mean ± SD relative luciferase activity in triplicate cultures and are representative of two independent experiments. (B) Inhibition of HCVpp-mediated luciferase expression by RO4475417 when added to cell culture media 1 h prior to and during HCVpp infection (−1 to 6 h, as described for panel A) or following a 6-h incubation with HCVpp (6 to 12 h). Results shown represent mean ± SE relative luciferase activity. (C) Inhibition of cap-dependent and HCV IRES-dependent translation following a 6-h exposure to increasing concentrations of RO4475417 (MKNK inhibitor). Cells were transfected with pRLHL, which expresses a dicistronic transcript containing the HCV IRES within the intercistronic space. Twenty-four h later (time point 0), cells were treated with RO4475417 for 6 h. Cells were lysed and luciferase activities measured at 48 h. Results shown represent RLuc (cap-dependent translation initiation) and FLuc (IRES-dependent) activities relative to those in the absence of the compound (100%) and are the means ± SD from triplicate cultures. (D) Kinetic analysis of recovery from RO4475417-mediated inhibition of cap-dependent translation. Cells were transfected with pCMV-GLuc, which expresses secreted GLuc, 24 h prior to drug exposure, as described for panel C. Supernatant fluids were harvested for GLuc assay and replaced with fresh medium at 6, 24, and 48 h. (E) Lack of an effect of a 6-h exposure to RO4475417 (5 μM) on cellular abundance of the HCV coreceptors CD81, SR-B1, claudin-1, occludin, and NPC1L1. Cells were treated with compound for 6 h starting at time point 0 and harvested for immunoblot assays at 6 and 24 h.

    Techniques Used: Inhibition, Luciferase, Infection, Activity Assay, Expressing, Cell Culture, Incubation, Transfection

    Related Articles

    Plasmid Preparation:

    Article Title: Replacement of a Thiourea with an Amidine Group in a Monofunctional Platinum-acridine Antitumor Agent. Effect on DNA Interactions, DNA Adduct Recognition and Repair
    Article Snippet: 20 000 kDa) was prepared and characterized as described previously., The plasmids, pUC19 (2686 bp) and pBR322 (4361 bp), were isolated according to standard procedures. .. Restriction endonucleases, plasmid DNA pCMV-GLuc (5764 bp), T4 DNA ligase and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). .. The synthetic oligodeoxyribonucleotides were purchased from VBC-GENOMICS (Vienna, Austria) or DNA Technology (Aarhus, Denmark).

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: To create pIR-CMVluc, these two PCR products were ligated using Mighty Cloning Kit (blunt end). .. The expression cassette including Gluc gene under CMV promoter control was amplified by PCR using pCMV-Gluc Control Plasmid (New England BioLabs Japan, Tokyo, Japan) as a template, primer9, and primer10. .. This PCR product was ligated with p3EIR to create pIR-CMVGluc. hEF1 promoter was amplified by PCR using pBLAST49-hHGF as a template, primer11, and primer12.

    Article Title: Interferon Kappa Inhibits Human Papillomavirus 31 Transcription by Inducing Sp100 Proteins
    Article Snippet: Cells were cotransfected with 100 ng of pGL18 URR or pGL31 URR reporter plasmid and 100 ng of the vector control (pSG5) or Sp100 A, -B, -C, or -HMG expression vector using the FuGENE HD reagent (Promega) and Opti-MEM (Life Technologies). .. Furthermore, 0.5 ng of pCMV-Gluc plasmid (New England BioLabs) was included as an internal control. .. Gaussia and firefly luciferase assays were carried out 48 h after transfection.

    Article Title: Conformation and recognition of DNA modified by a new antitumor dinuclear PtII complex resistant to decomposition by sulfur nucleophiles
    Article Snippet: Plasmids pSP73 [2464 base pairs (bp)], pUC19 (2 686 bp), and pBR322 (4361 bp) were isolated according to standard procedures. .. The Klenow fragment from DNA polymerase I (exonuclease minus, mutated to remove the 3′ → 5′ proofreading domain), restriction endonucleases EcoRI, SspI, and XbaI, Circum Vent™ Thermal Cycle Sequencing Kit with Vent(exo− ) DNA polymerase, and plasmid DNA pCMV-GLuc (5764 bp) were purchased from New England Biolabs (Beverly, MA). .. HeLaScribe® Nuclear Extract in vitro Transcription system kit was from Promega (Mannheim, Germany).

    Article Title: Characterization of the Human Papillomavirus 16 E8 Promoter
    Article Snippet: Cells were transfected with reporter plasmids alone or together with pSG 16 E1 co, pSG 16 E2 (or pSG16 E2 R37A/I73A), or pSG 16 E8^E2C (or 16 E8^E2C KWK mt) expression constructs or the empty vector pSG5 as indicated in the figure legends using Fugene HD (Promega) and Opti-MEM (Life Technologies). .. In addition, the pCMV-Gluc plasmid (New England BioLabs) was cotransfected as an internal control. .. Gaussia and firefly luciferase assays were carried out 48 h after transfection.

    Expressing:

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: To create pIR-CMVluc, these two PCR products were ligated using Mighty Cloning Kit (blunt end). .. The expression cassette including Gluc gene under CMV promoter control was amplified by PCR using pCMV-Gluc Control Plasmid (New England BioLabs Japan, Tokyo, Japan) as a template, primer9, and primer10. .. This PCR product was ligated with p3EIR to create pIR-CMVGluc. hEF1 promoter was amplified by PCR using pBLAST49-hHGF as a template, primer11, and primer12.

    Amplification:

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: To create pIR-CMVluc, these two PCR products were ligated using Mighty Cloning Kit (blunt end). .. The expression cassette including Gluc gene under CMV promoter control was amplified by PCR using pCMV-Gluc Control Plasmid (New England BioLabs Japan, Tokyo, Japan) as a template, primer9, and primer10. .. This PCR product was ligated with p3EIR to create pIR-CMVGluc. hEF1 promoter was amplified by PCR using pBLAST49-hHGF as a template, primer11, and primer12.

    Polymerase Chain Reaction:

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: To create pIR-CMVluc, these two PCR products were ligated using Mighty Cloning Kit (blunt end). .. The expression cassette including Gluc gene under CMV promoter control was amplified by PCR using pCMV-Gluc Control Plasmid (New England BioLabs Japan, Tokyo, Japan) as a template, primer9, and primer10. .. This PCR product was ligated with p3EIR to create pIR-CMVGluc. hEF1 promoter was amplified by PCR using pBLAST49-hHGF as a template, primer11, and primer12.

    Activity Assay:

    Article Title: A survivin-driven, tumor-activatable minicircle system for prostate cancer theranostics
    Article Snippet: Separately seeded cells were transfected with pCMV-GLuc plasmids (1 μg, NEB), and GLuc activity was measured using the same kit. .. GLuc activity from pSurvivin-GLuc-MCs was normalized to GLuc activity from pCMV-GLuc plasmids. .. To compare expression from MCs with their PP counterparts, PC3MLN4 cells were seeded at 5 × 104 cells/well in a 24-well plate.

    Sequencing:

    Article Title: Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase
    Article Snippet: .. pCMV-GLuc, containing the GLuc coding sequence, was purchased from New England BioLabs (NEB). .. The pPV-GLuc plasmid was constructed from pT7PVM, which contains the cDNA of type 1 poliovirus [PV1(M)].

    Article Title: Conformation and recognition of DNA modified by a new antitumor dinuclear PtII complex resistant to decomposition by sulfur nucleophiles
    Article Snippet: Plasmids pSP73 [2464 base pairs (bp)], pUC19 (2 686 bp), and pBR322 (4361 bp) were isolated according to standard procedures. .. The Klenow fragment from DNA polymerase I (exonuclease minus, mutated to remove the 3′ → 5′ proofreading domain), restriction endonucleases EcoRI, SspI, and XbaI, Circum Vent™ Thermal Cycle Sequencing Kit with Vent(exo− ) DNA polymerase, and plasmid DNA pCMV-GLuc (5764 bp) were purchased from New England Biolabs (Beverly, MA). .. HeLaScribe® Nuclear Extract in vitro Transcription system kit was from Promega (Mannheim, Germany).

    Incubation:

    Article Title: Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein
    Article Snippet: .. At 16 h posttransfection, the culture medium was changed, and the cells were incubated for 4 h. As a control, cells transfected with 25 ng pCMV-GLuc and 300 ng pcDNA3.1 were treated with 1 μg/ml BFA during this 4-h secretion period. .. Luciferase activity was measured in the supernatant and cell lysates using a Renilla luciferase assay system kit from Promega as recommended by the manufacturer.

    Transfection:

    Article Title: Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein
    Article Snippet: .. At 16 h posttransfection, the culture medium was changed, and the cells were incubated for 4 h. As a control, cells transfected with 25 ng pCMV-GLuc and 300 ng pcDNA3.1 were treated with 1 μg/ml BFA during this 4-h secretion period. .. Luciferase activity was measured in the supernatant and cell lysates using a Renilla luciferase assay system kit from Promega as recommended by the manufacturer.

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    New England Biolabs pcmv gluc
    Effect of brefeldin A on <t>GLuc</t> secretion. (A) Schematic representation of the GLuc-expressing reporter construct <t>pCMV-GLuc.</t> (B) Inhibition of GLuc transport in the presence of the indicated concentrations of BFA. HeLa cells were transfected with a GLuc-expressing vector. At 6 h posttransfection, cells were treated with different amounts of BFA. Samples taken from growth medium and lysates were assayed for GLuc activity at 16 h after addition of BFA.
    Pcmv Gluc, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv gluc/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcmv gluc - by Bioz Stars, 2021-06
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    Effect of brefeldin A on GLuc secretion. (A) Schematic representation of the GLuc-expressing reporter construct pCMV-GLuc. (B) Inhibition of GLuc transport in the presence of the indicated concentrations of BFA. HeLa cells were transfected with a GLuc-expressing vector. At 6 h posttransfection, cells were treated with different amounts of BFA. Samples taken from growth medium and lysates were assayed for GLuc activity at 16 h after addition of BFA.

    Journal: Journal of Virology

    Article Title: Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

    doi: 10.1128/JVI.05871-11

    Figure Lengend Snippet: Effect of brefeldin A on GLuc secretion. (A) Schematic representation of the GLuc-expressing reporter construct pCMV-GLuc. (B) Inhibition of GLuc transport in the presence of the indicated concentrations of BFA. HeLa cells were transfected with a GLuc-expressing vector. At 6 h posttransfection, cells were treated with different amounts of BFA. Samples taken from growth medium and lysates were assayed for GLuc activity at 16 h after addition of BFA.

    Article Snippet: pCMV-GLuc, containing the GLuc coding sequence, was purchased from New England BioLabs (NEB).

    Techniques: Expressing, Construct, Inhibition, Transfection, Plasmid Preparation, Activity Assay

    The eEF1A inhibitor didemnin B blocks HIV-1 reverse transcription. ( A ) Jurkat cells transfected with pCMV-Gluc 2 were treated post-transfection with media containing didemnin B (Did B) or cycloheximide (CHX) at concentrations as indicated. The levels of secreted Gaussia luciferase in the culture supernatants were measured 8 h post-treatment. ( B and C ) Jurkat cells were incubated with subnanomolar concentrations of Did B and CHX as indicated and then infected with HIV-1 at 4°C for 2 h and 37°C for 4 h. The total cytoplasmic DNA was collected and the levels of HIV-1 early ( B ) and late ( C ) reverse transcription products were measured by qPCR. ( D ) Reverse transcription efficiencies were calculated as the ratio of late to early viral DNA, expressed as a percentage. ( E ) The levels of HIV-1 RNA in cells treated with didemnin B (Did B) and cycloheximide (CHX). Jurkat cells were incubated with 100 nM nevirapine and concentrations of Did B and CHX as indicated at 37°C for 2 h followed by HIV-1 infection. The HIV-1 was incubated with cells at 4°C for 2 h and then at 37°C for 4 h. Heat inactivated virus was used as a negative control. The HIV-1 RNA was extracted from the cells and detected by RT-qPCR. A direct qPCR without RT from the RNA samples was performed to monitor for DNA contamination. All columns represent a mean value and standard deviation from at least three independent experiments. ** = p

    Journal: PLoS Pathogens

    Article Title: Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target

    doi: 10.1371/journal.ppat.1005289

    Figure Lengend Snippet: The eEF1A inhibitor didemnin B blocks HIV-1 reverse transcription. ( A ) Jurkat cells transfected with pCMV-Gluc 2 were treated post-transfection with media containing didemnin B (Did B) or cycloheximide (CHX) at concentrations as indicated. The levels of secreted Gaussia luciferase in the culture supernatants were measured 8 h post-treatment. ( B and C ) Jurkat cells were incubated with subnanomolar concentrations of Did B and CHX as indicated and then infected with HIV-1 at 4°C for 2 h and 37°C for 4 h. The total cytoplasmic DNA was collected and the levels of HIV-1 early ( B ) and late ( C ) reverse transcription products were measured by qPCR. ( D ) Reverse transcription efficiencies were calculated as the ratio of late to early viral DNA, expressed as a percentage. ( E ) The levels of HIV-1 RNA in cells treated with didemnin B (Did B) and cycloheximide (CHX). Jurkat cells were incubated with 100 nM nevirapine and concentrations of Did B and CHX as indicated at 37°C for 2 h followed by HIV-1 infection. The HIV-1 was incubated with cells at 4°C for 2 h and then at 37°C for 4 h. Heat inactivated virus was used as a negative control. The HIV-1 RNA was extracted from the cells and detected by RT-qPCR. A direct qPCR without RT from the RNA samples was performed to monitor for DNA contamination. All columns represent a mean value and standard deviation from at least three independent experiments. ** = p

    Article Snippet: The secreted Gaussia luciferase expression construct, pCMV-Gluc 2 was purchased from NEB (NEB, USA).

    Techniques: Transfection, Luciferase, Incubation, Infection, Real-time Polymerase Chain Reaction, Negative Control, Quantitative RT-PCR, Standard Deviation

    Characterization of secreted reporter expression in vitro (A and B) Vector map of the pSurv-GLuc-PP expression cassette (A) with agarose gel electrophoresis (B) to confirm proper production of the PP (6.9 kb) and MC (2.9 kb). (C) Western blot for cellular survivin expression in PCa and primary prostate epithelial cells (n = 3). Band intensities were quantified using ImageJ and are shown relative to GAPDH. (D) GLuc bioluminescence signal above background from PCa cells transfected with pSurv-GLuc-MCs (n = 5). (E–G) GLuc activity in supernatant from cell lines transfected with (E) pSurv-GLuc-MCs or (F) pCMV-GLuc plasmids on day 2 post-transfection with (G) normalized values (n = 3). (H) Groups designated by different letters are significantly different (p

    Journal: Molecular Therapy Oncolytics

    Article Title: A survivin-driven, tumor-activatable minicircle system for prostate cancer theranostics

    doi: 10.1016/j.omto.2021.01.007

    Figure Lengend Snippet: Characterization of secreted reporter expression in vitro (A and B) Vector map of the pSurv-GLuc-PP expression cassette (A) with agarose gel electrophoresis (B) to confirm proper production of the PP (6.9 kb) and MC (2.9 kb). (C) Western blot for cellular survivin expression in PCa and primary prostate epithelial cells (n = 3). Band intensities were quantified using ImageJ and are shown relative to GAPDH. (D) GLuc bioluminescence signal above background from PCa cells transfected with pSurv-GLuc-MCs (n = 5). (E–G) GLuc activity in supernatant from cell lines transfected with (E) pSurv-GLuc-MCs or (F) pCMV-GLuc plasmids on day 2 post-transfection with (G) normalized values (n = 3). (H) Groups designated by different letters are significantly different (p

    Article Snippet: GLuc activity from pSurvivin-GLuc-MCs was normalized to GLuc activity from pCMV-GLuc plasmids.

    Techniques: Expressing, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis, Western Blot, Transfection, Activity Assay

    Sp100B, -C, and -HMG inhibit the HPV18 and HPV31 URR. CIN612-9E cells were transfected with 0.5 ng of pCMV-Gluc, 100 ng of Sp100B, -C, or -HMG or empty vector (vec) expression plasmid, and 100 ng of HPV18 URR or 31 URR reporter plasmid. Values are presented

    Journal: Journal of Virology

    Article Title: Interferon Kappa Inhibits Human Papillomavirus 31 Transcription by Inducing Sp100 Proteins

    doi: 10.1128/JVI.02137-15

    Figure Lengend Snippet: Sp100B, -C, and -HMG inhibit the HPV18 and HPV31 URR. CIN612-9E cells were transfected with 0.5 ng of pCMV-Gluc, 100 ng of Sp100B, -C, or -HMG or empty vector (vec) expression plasmid, and 100 ng of HPV18 URR or 31 URR reporter plasmid. Values are presented

    Article Snippet: Furthermore, 0.5 ng of pCMV-Gluc plasmid (New England BioLabs) was included as an internal control.

    Techniques: Transfection, Plasmid Preparation, Expressing