pcmv gluc plasmid  (New England Biolabs)


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    Name:
    pCMV GLuc Control Plasmid
    Description:
    pCMV GLuc Control Plasmid 20 ug
    Catalog Number:
    n8081s
    Price:
    313
    Size:
    20 ug
    Category:
    Cellular Biology
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    Structured Review

    New England Biolabs pcmv gluc plasmid
    pCMV GLuc Control Plasmid
    pCMV GLuc Control Plasmid 20 ug
    https://www.bioz.com/result/pcmv gluc plasmid/product/New England Biolabs
    Average 95 stars, based on 88 article reviews
    Price from $9.99 to $1999.99
    pcmv gluc plasmid - by Bioz Stars, 2020-05
    95/100 stars

    Images

    1) Product Images from "The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16"

    Article Title: The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16

    Journal: Journal of Virology

    doi: 10.1128/JVI.02296-13

    Characterization of HPV16 E8^E2C wt and KWK mutant proteins. (A) HeLa or RTS3b cells were cotransfected with pC18-Sp1-luc (100 ng), 10 ng of pSG5 (vec), pSG 16 E8^E2C, or pSG 16 E8^E2C KWK mt and 0.1 ng of pCMV-Gluc. (B) HeLa or RTS3b cells were cotransfected
    Figure Legend Snippet: Characterization of HPV16 E8^E2C wt and KWK mutant proteins. (A) HeLa or RTS3b cells were cotransfected with pC18-Sp1-luc (100 ng), 10 ng of pSG5 (vec), pSG 16 E8^E2C, or pSG 16 E8^E2C KWK mt and 0.1 ng of pCMV-Gluc. (B) HeLa or RTS3b cells were cotransfected

    Techniques Used: Mutagenesis

    2) Product Images from "A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication"

    Article Title: A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000216

    GBF1 sequence determines BFA resistance of cells and virus. A. Sequencing chromatograms of GBF1 Sec7 domain from Vero and BER-40 cells. Results from analyses with three different primers are shown. B. GBF1 A795E mutant confers BFA resistance to HeLa cells. Cells were transfected with either a control vector, vector expressing wild type YFP-GBF1 fusion or vector expressing YFP-GBF1 A795E mutant fusion, and subsequent cell growth in the presence of BFA was measured by a luminescent cell viability assay. C. Expression of GBF1 A795E mutant rescues protein secretion in the presence of BFA. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with either control plasmid or with vectors expressing wild type GBF1-YFP or GBF1 A795E-YFP fusions. The amount of secreted protein observed in each sample without BFA was defined as 100%. D. GBF1 A795E mutant rescues replication of poliovirus in the presence of BFA. A polio Renilla luciferase replicon was introduced in HeLa cells previously transfected with vectors expressing wt YFP-GBF1 fusion, YFP-GBF1 A795E mutant or with an empty vector. BFA was added where indicated at 1 µg/ml concentration at the time of replicon transfection, and polio RNA replication was measured by luciferase assay. Expression of the GBF1 proteins was measured by Western blot; calnexin staining was used as a loading control (panel D, right).
    Figure Legend Snippet: GBF1 sequence determines BFA resistance of cells and virus. A. Sequencing chromatograms of GBF1 Sec7 domain from Vero and BER-40 cells. Results from analyses with three different primers are shown. B. GBF1 A795E mutant confers BFA resistance to HeLa cells. Cells were transfected with either a control vector, vector expressing wild type YFP-GBF1 fusion or vector expressing YFP-GBF1 A795E mutant fusion, and subsequent cell growth in the presence of BFA was measured by a luminescent cell viability assay. C. Expression of GBF1 A795E mutant rescues protein secretion in the presence of BFA. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with either control plasmid or with vectors expressing wild type GBF1-YFP or GBF1 A795E-YFP fusions. The amount of secreted protein observed in each sample without BFA was defined as 100%. D. GBF1 A795E mutant rescues replication of poliovirus in the presence of BFA. A polio Renilla luciferase replicon was introduced in HeLa cells previously transfected with vectors expressing wt YFP-GBF1 fusion, YFP-GBF1 A795E mutant or with an empty vector. BFA was added where indicated at 1 µg/ml concentration at the time of replicon transfection, and polio RNA replication was measured by luciferase assay. Expression of the GBF1 proteins was measured by Western blot; calnexin staining was used as a loading control (panel D, right).

    Techniques Used: Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Cell Viability Assay, Luciferase, Concentration Assay, Western Blot, Staining

    3) Product Images from "piggyBac Transposon-mediated Long-term Gene Expression in Mice"

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice

    Journal: Molecular Therapy

    doi: 10.1038/mt.2009.302

    Sustained Gluc expression in vivo . Expression time course of Gluc from PB -based ( a,c ) or conventional ( b ) pDNA. 25 µg pIR-CMVGluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( a ). 25 µg pCMV-Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( b ). 25 µg pIR-EF1Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( c ). Blood samples were collected at the indicated time points, and Gluc activities in serum were measured. Each value represents mean ± SD ( n = 4–6). RLU, relative light unit.
    Figure Legend Snippet: Sustained Gluc expression in vivo . Expression time course of Gluc from PB -based ( a,c ) or conventional ( b ) pDNA. 25 µg pIR-CMVGluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( a ). 25 µg pCMV-Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( b ). 25 µg pIR-EF1Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( c ). Blood samples were collected at the indicated time points, and Gluc activities in serum were measured. Each value represents mean ± SD ( n = 4–6). RLU, relative light unit.

    Techniques Used: Expressing, In Vivo, Injection

    4) Product Images from "GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication ▿"

    Article Title: GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01244-09

    BFA-resistant GBF1 mutants M832L and A795E differ in their abilities to rescue protein secretion and cell viability in the presence of BFA. (A) HeLa cells were cotransfected with plasmid pCMV-Gluc expressing secreted Gaussia luciferase and either a control
    Figure Legend Snippet: BFA-resistant GBF1 mutants M832L and A795E differ in their abilities to rescue protein secretion and cell viability in the presence of BFA. (A) HeLa cells were cotransfected with plasmid pCMV-Gluc expressing secreted Gaussia luciferase and either a control

    Techniques Used: Plasmid Preparation, Expressing, Luciferase

    5) Product Images from "Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1"

    Article Title: Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2010.01482.x

    Truncated GBF1 mutants do not rescue cellular secretion from BFA inhibition A. Schematic map of GBF1 domain organization and of truncated mutants used in this study. Numbers in parenthesis indicate GBF1 amino acids. B. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with vectors expressing full length YFP-GBF1A795E, truncated GBF1 fusions, or an empty vector (control). Cells were incubated for 5 h in the presence of 1 μg/ml BFA or corresponding amount of DMSO. The luciferase activity observed in each sample without BFA was defined as 100%. Western blots show expression of GBF1 species detected with anti-GFP antibodies. Actin immunoblots are shown as a loading control.
    Figure Legend Snippet: Truncated GBF1 mutants do not rescue cellular secretion from BFA inhibition A. Schematic map of GBF1 domain organization and of truncated mutants used in this study. Numbers in parenthesis indicate GBF1 amino acids. B. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with vectors expressing full length YFP-GBF1A795E, truncated GBF1 fusions, or an empty vector (control). Cells were incubated for 5 h in the presence of 1 μg/ml BFA or corresponding amount of DMSO. The luciferase activity observed in each sample without BFA was defined as 100%. Western blots show expression of GBF1 species detected with anti-GFP antibodies. Actin immunoblots are shown as a loading control.

    Techniques Used: Inhibition, Transfection, Plasmid Preparation, Expressing, Luciferase, Incubation, Activity Assay, Western Blot

    6) Product Images from "Secreted Luciferase for In Vivo Evaluation of Systemic Protein Delivery in Mice"

    Article Title: Secreted Luciferase for In Vivo Evaluation of Systemic Protein Delivery in Mice

    Journal: Molecular biotechnology

    doi: 10.1007/s12033-012-9519-6

    In vivo Gluc expression and secretion. Gluc activities were determined in liver homogenates and EDTA-blood of C57 mice 24 hr after HTV injection with either pCMV-Gluc or phAAT-Gluc plasmids. Luciferase activities were normalized by subtracting background
    Figure Legend Snippet: In vivo Gluc expression and secretion. Gluc activities were determined in liver homogenates and EDTA-blood of C57 mice 24 hr after HTV injection with either pCMV-Gluc or phAAT-Gluc plasmids. Luciferase activities were normalized by subtracting background

    Techniques Used: In Vivo, Expressing, Mouse Assay, Injection, Luciferase

    Time-dependent Gluc activities in the circulation after HTV injection of pCMV-Gluc plasmid. Blood samples were collected and analyzed at various time points from injected mice (n=4) of either the immune-competent C57 strain or the immune-deficient SCID-β2
    Figure Legend Snippet: Time-dependent Gluc activities in the circulation after HTV injection of pCMV-Gluc plasmid. Blood samples were collected and analyzed at various time points from injected mice (n=4) of either the immune-competent C57 strain or the immune-deficient SCID-β2

    Techniques Used: Injection, Plasmid Preparation, Mouse Assay

    Bio-imaging of Gluc expression and correlation of in vivo bioluminescence signals with blood luciferase activities. Bioluminescence imaging analysis was performed in mice 1–2 days after HTV injection of either saline (mock-injected, n=3) or pCMV-Gluc
    Figure Legend Snippet: Bio-imaging of Gluc expression and correlation of in vivo bioluminescence signals with blood luciferase activities. Bioluminescence imaging analysis was performed in mice 1–2 days after HTV injection of either saline (mock-injected, n=3) or pCMV-Gluc

    Techniques Used: Imaging, Expressing, In Vivo, Luciferase, Mouse Assay, Injection

    7) 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

    8) Product Images from "Interferon Kappa Inhibits Human Papillomavirus 31 Transcription by Inducing Sp100 Proteins"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.02137-15

    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
    Figure Legend 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

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    9) Product Images from "Peptide-Targeted Polyplexes for Aerosol-Mediated Gene Delivery to CD49f-Overexpressing Tumor Lesions in Lung"

    Article Title: Peptide-Targeted Polyplexes for Aerosol-Mediated Gene Delivery to CD49f-Overexpressing Tumor Lesions in Lung

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.10.009

    Transfection Efficiency of Polyplexes on MDA-MB-231 and 4T1-iRFP720 Breast Cancer Cells (A and B) MDA-MB-231 (A) and 4T1-iRFP720 cells (B) were seeded in 96-well plates and transfected with pCMV-Gluc polyplexes at indicated pDNA concentrations. Transfections were conducted in basal cell culture medium for 4 h followed by exchanging the supernatant with fetal calf serum (FCS)-supplemented medium. Gluc was quantified in the supernatant 24 h after transfection, and RLU values were normalized on total count of live cells (MDA-MB-231) or on protein content (4T1-iRFP720). Mean values are from two independent experiments with n = 3/experiment + SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Mann-Whitney).
    Figure Legend Snippet: Transfection Efficiency of Polyplexes on MDA-MB-231 and 4T1-iRFP720 Breast Cancer Cells (A and B) MDA-MB-231 (A) and 4T1-iRFP720 cells (B) were seeded in 96-well plates and transfected with pCMV-Gluc polyplexes at indicated pDNA concentrations. Transfections were conducted in basal cell culture medium for 4 h followed by exchanging the supernatant with fetal calf serum (FCS)-supplemented medium. Gluc was quantified in the supernatant 24 h after transfection, and RLU values were normalized on total count of live cells (MDA-MB-231) or on protein content (4T1-iRFP720). Mean values are from two independent experiments with n = 3/experiment + SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Mann-Whitney).

    Techniques Used: Transfection, Multiple Displacement Amplification, Cell Culture, MANN-WHITNEY

    10) 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

    11) Product Images from "Characterization of the Human Papillomavirus 16 E8 Promoter"

    Article Title: Characterization of the Human Papillomavirus 16 E8 Promoter

    Journal: Journal of Virology

    doi: 10.1128/JVI.00616-15

    Regulation of the E8 promoter by the URR and E8^E2C. (A, B) RTS3b or NHK cells were transfected with 0.5 ng of pCMV-Gluc and 50 or 300 ng of the indicated reporter plasmids, respectively. Values are presented as the ratios of fluc to gluc activities,
    Figure Legend Snippet: Regulation of the E8 promoter by the URR and E8^E2C. (A, B) RTS3b or NHK cells were transfected with 0.5 ng of pCMV-Gluc and 50 or 300 ng of the indicated reporter plasmids, respectively. Values are presented as the ratios of fluc to gluc activities,

    Techniques Used: Transfection

    Regulation of the E8 promoter by E1 and E2. RTS3b cells were cotransfected with 0.5 ng of pCMV-Gluc, the indicated reporter plasmids (50 ng), and the empty vector (−) or a combination of 100 ng pSG16 E1co and 10 ng pSG16 E2 (+).Values are presented
    Figure Legend Snippet: Regulation of the E8 promoter by E1 and E2. RTS3b cells were cotransfected with 0.5 ng of pCMV-Gluc, the indicated reporter plasmids (50 ng), and the empty vector (−) or a combination of 100 ng pSG16 E1co and 10 ng pSG16 E2 (+).Values are presented

    Techniques Used: Plasmid Preparation

    Two regions are required for E8 promoter activity in keratinocytes. RTS3b or NHK cells were transfected with 0.5 ng of pCMV-Gluc and 50 or 300 ng of the indicated reporter plasmids, respectively. (Upper panels) Values are presented as the ratios of firefly
    Figure Legend Snippet: Two regions are required for E8 promoter activity in keratinocytes. RTS3b or NHK cells were transfected with 0.5 ng of pCMV-Gluc and 50 or 300 ng of the indicated reporter plasmids, respectively. (Upper panels) Values are presented as the ratios of firefly

    Techniques Used: Activity Assay, Transfection

    Influence of a BRD4-binding-deficient E2 mutant on E8 promoter activity. (A) RTS3b cells were cotransfected with 0.5 ng of pCMV-Gluc, 50 ng reporter, and 30 ng of the empty vector (vec), pSG16 E2 (E2), or pSG16 E2 R37A/I73A (E2 mt). Values are presented
    Figure Legend Snippet: Influence of a BRD4-binding-deficient E2 mutant on E8 promoter activity. (A) RTS3b cells were cotransfected with 0.5 ng of pCMV-Gluc, 50 ng reporter, and 30 ng of the empty vector (vec), pSG16 E2 (E2), or pSG16 E2 R37A/I73A (E2 mt). Values are presented

    Techniques Used: Binding Assay, Mutagenesis, Activity Assay, Plasmid Preparation

    12) Product Images from "Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8^E2 Protein"

    Article Title: Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8^E2 Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01743-17

    E2 S266A does not restore repression activity of E8^E2 S78A. RTS3b cells were transfected with 0.5 ng of pCMV-Gluc, 50 ng of pGL31URR reporter plasmid, expression vectors for HPV31 E1 (100 ng), and the indicated expression vectors for HPV31 E2 (10 ng) and E8^E2 (10 ng). Differences in the amounts of DNA were adjusted with the empty expression vectors (pSG5). Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the SEM from four independent experiments performed in duplicate.
    Figure Legend Snippet: E2 S266A does not restore repression activity of E8^E2 S78A. RTS3b cells were transfected with 0.5 ng of pCMV-Gluc, 50 ng of pGL31URR reporter plasmid, expression vectors for HPV31 E1 (100 ng), and the indicated expression vectors for HPV31 E2 (10 ng) and E8^E2 (10 ng). Differences in the amounts of DNA were adjusted with the empty expression vectors (pSG5). Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the SEM from four independent experiments performed in duplicate.

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase

    Phosphorylation of E8^E2 S78 is required for transcriptional repression of reporter plasmids. (A) HeLa cells were transfected with 100 ng of the pC18-Sp1-luc firefly luciferase reporter, 10 ng of the empty expression vector (pSG5), or the expression vectors for wild-type or mutant HPV31 E8^E2 (left graph) or HPV31 E2 (right graph) and 0.5 ng pCMV-Gluc as an internal control. (B) NHK-HPV31 wild-type cells were transfected with 300 ng pC18-Sp1-luc, 30 ng of the empty vector, or the indicated HPV31 E8^E2 expression vectors (wild type or mutants) and 0.5 ng pCMV-Gluc. Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the standard error of the mean (SEM) from at least seven independent experiments (HeLa) or three independent experiments (NHK-HPV31 WT) performed in duplicate. Statistical significance was determined with a one-way ANOVA and Dunnett's multiple-comparison test: *, P
    Figure Legend Snippet: Phosphorylation of E8^E2 S78 is required for transcriptional repression of reporter plasmids. (A) HeLa cells were transfected with 100 ng of the pC18-Sp1-luc firefly luciferase reporter, 10 ng of the empty expression vector (pSG5), or the expression vectors for wild-type or mutant HPV31 E8^E2 (left graph) or HPV31 E2 (right graph) and 0.5 ng pCMV-Gluc as an internal control. (B) NHK-HPV31 wild-type cells were transfected with 300 ng pC18-Sp1-luc, 30 ng of the empty vector, or the indicated HPV31 E8^E2 expression vectors (wild type or mutants) and 0.5 ng pCMV-Gluc. Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the standard error of the mean (SEM) from at least seven independent experiments (HeLa) or three independent experiments (NHK-HPV31 WT) performed in duplicate. Statistical significance was determined with a one-way ANOVA and Dunnett's multiple-comparison test: *, P

    Techniques Used: Transfection, Luciferase, Expressing, Plasmid Preparation, Mutagenesis

    Phosphorylation of E8^E2 S78 is required for repression of replication in a reporter-based replication assay. RTS3b cells were transfected with 0.5 ng of pCMV-Gluc, 50 ng of a reporter plasmid containing the HPV31 URR and the viral early promoter driving the firefly luciferase gene (pGL31URR), and expression vectors for HPV31 E1 (100 ng), E2 (10 ng), and wild-type or mutant E8^E2 (10 ng). Differences in the amounts of DNA were adjusted with the empty expression vectors (pSG5). Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the SEM from five independent experiments performed in duplicate. Statistical significance was determined with a one-way ANOVA and Dunnett's multiple-comparison test: *, P
    Figure Legend Snippet: Phosphorylation of E8^E2 S78 is required for repression of replication in a reporter-based replication assay. RTS3b cells were transfected with 0.5 ng of pCMV-Gluc, 50 ng of a reporter plasmid containing the HPV31 URR and the viral early promoter driving the firefly luciferase gene (pGL31URR), and expression vectors for HPV31 E1 (100 ng), E2 (10 ng), and wild-type or mutant E8^E2 (10 ng). Differences in the amounts of DNA were adjusted with the empty expression vectors (pSG5). Values are presented as the ratio of firefly luciferase (Fluc) to Gaussia luciferase (Gluc) activities. Error bars indicate the SEM from five independent experiments performed in duplicate. Statistical significance was determined with a one-way ANOVA and Dunnett's multiple-comparison test: *, P

    Techniques Used: Transfection, Plasmid Preparation, Luciferase, Expressing, Mutagenesis

    13) 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

    Related Articles

    Amplification:

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: .. 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.

    Luciferase:

    Article Title: GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication ▿
    Article Snippet: .. Plasmid pCMV-Gluc, which expresses secreted Gaussia luciferase, was purchased from New England Biolabs. .. Confluent monolayers of BGM or HeLa cells were infected with virus for 30 min at 37°C at a multiplicity of infection (MOI) of 10 TCID50 per cell.

    Article Title: Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1
    Article Snippet: .. Plasmid pCMV-Gluc for expression of secreted Gaussia luciferase was from New England Biolabs. .. Plasmid pXpa-RenR coding for a poliovirus replicon with the Renilla luciferase gene substituted for the capsid coding sequence was described previously ( ).

    Derivative Assay:

    Article Title: Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase
    Article Snippet: .. The nontranslated region and the poly(A) signal at the 3′ end were derived from plasmid pCMV-GLuc (NEB). ..

    Expressing:

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: .. 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: Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1
    Article Snippet: .. Plasmid pCMV-Gluc for expression of secreted Gaussia luciferase was from New England Biolabs. .. Plasmid pXpa-RenR coding for a poliovirus replicon with the Renilla luciferase gene substituted for the capsid coding sequence was described previously ( ).

    Polymerase Chain Reaction:

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: .. 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.

    Plasmid Preparation:

    Article Title: Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase
    Article Snippet: .. The nontranslated region and the poly(A) signal at the 3′ end were derived from plasmid pCMV-GLuc (NEB). ..

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice
    Article Snippet: .. 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: Secreted Luciferase for In Vivo Evaluation of Systemic Protein Delivery in Mice
    Article Snippet: .. The pCMV-Gluc plasmid was obtained from New England BioLabs (Ipswich, MA). .. The phAAT-Gluc plasmid was constructed using the pCMV-Gluc and pBS-HCRHPI-A plasmids.

    Article Title: The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16
    Article Snippet: .. Furthermore, 0.1 ng of pCMV-Gluc plasmid (New England BioLabs) was cotransfected as an internal control. .. Gaussia and firefly luciferase assays were carried out 48 h after transfection.

    Article Title: Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1
    Article Snippet: .. Plasmid pCMV-Gluc for expression of secreted Gaussia luciferase was from New England Biolabs. .. Plasmid pXpa-RenR coding for a poliovirus replicon with the Renilla luciferase gene substituted for the capsid coding sequence was described previously ( ).

    Article Title: A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication
    Article Snippet: .. Plasmid pCMV-Gluc used for the secretion rescue experiment was purchased from New England Biolabs. .. Antibodies Rabbit polyclonal anti-GBF1 antibodies were a gift from N. Altan-Bonnet, Rutgers University, New Jersey.

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    New England Biolabs pcmv gluc plasmid
    Characterization of HPV16 E8^E2C wt and KWK mutant proteins. (A) HeLa or RTS3b cells were cotransfected with pC18-Sp1-luc (100 ng), 10 ng of pSG5 (vec), pSG 16 E8^E2C, or pSG 16 E8^E2C KWK mt and 0.1 ng of <t>pCMV-Gluc.</t> (B) HeLa or RTS3b cells were cotransfected
    Pcmv Gluc Plasmid, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of HPV16 E8^E2C wt and KWK mutant proteins. (A) HeLa or RTS3b cells were cotransfected with pC18-Sp1-luc (100 ng), 10 ng of pSG5 (vec), pSG 16 E8^E2C, or pSG 16 E8^E2C KWK mt and 0.1 ng of pCMV-Gluc. (B) HeLa or RTS3b cells were cotransfected

    Journal: Journal of Virology

    Article Title: The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16

    doi: 10.1128/JVI.02296-13

    Figure Lengend Snippet: Characterization of HPV16 E8^E2C wt and KWK mutant proteins. (A) HeLa or RTS3b cells were cotransfected with pC18-Sp1-luc (100 ng), 10 ng of pSG5 (vec), pSG 16 E8^E2C, or pSG 16 E8^E2C KWK mt and 0.1 ng of pCMV-Gluc. (B) HeLa or RTS3b cells were cotransfected

    Article Snippet: Furthermore, 0.1 ng of pCMV-Gluc plasmid (New England BioLabs) was cotransfected as an internal control.

    Techniques: Mutagenesis

    GBF1 sequence determines BFA resistance of cells and virus. A. Sequencing chromatograms of GBF1 Sec7 domain from Vero and BER-40 cells. Results from analyses with three different primers are shown. B. GBF1 A795E mutant confers BFA resistance to HeLa cells. Cells were transfected with either a control vector, vector expressing wild type YFP-GBF1 fusion or vector expressing YFP-GBF1 A795E mutant fusion, and subsequent cell growth in the presence of BFA was measured by a luminescent cell viability assay. C. Expression of GBF1 A795E mutant rescues protein secretion in the presence of BFA. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with either control plasmid or with vectors expressing wild type GBF1-YFP or GBF1 A795E-YFP fusions. The amount of secreted protein observed in each sample without BFA was defined as 100%. D. GBF1 A795E mutant rescues replication of poliovirus in the presence of BFA. A polio Renilla luciferase replicon was introduced in HeLa cells previously transfected with vectors expressing wt YFP-GBF1 fusion, YFP-GBF1 A795E mutant or with an empty vector. BFA was added where indicated at 1 µg/ml concentration at the time of replicon transfection, and polio RNA replication was measured by luciferase assay. Expression of the GBF1 proteins was measured by Western blot; calnexin staining was used as a loading control (panel D, right).

    Journal: PLoS Pathogens

    Article Title: A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication

    doi: 10.1371/journal.ppat.1000216

    Figure Lengend Snippet: GBF1 sequence determines BFA resistance of cells and virus. A. Sequencing chromatograms of GBF1 Sec7 domain from Vero and BER-40 cells. Results from analyses with three different primers are shown. B. GBF1 A795E mutant confers BFA resistance to HeLa cells. Cells were transfected with either a control vector, vector expressing wild type YFP-GBF1 fusion or vector expressing YFP-GBF1 A795E mutant fusion, and subsequent cell growth in the presence of BFA was measured by a luminescent cell viability assay. C. Expression of GBF1 A795E mutant rescues protein secretion in the presence of BFA. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with either control plasmid or with vectors expressing wild type GBF1-YFP or GBF1 A795E-YFP fusions. The amount of secreted protein observed in each sample without BFA was defined as 100%. D. GBF1 A795E mutant rescues replication of poliovirus in the presence of BFA. A polio Renilla luciferase replicon was introduced in HeLa cells previously transfected with vectors expressing wt YFP-GBF1 fusion, YFP-GBF1 A795E mutant or with an empty vector. BFA was added where indicated at 1 µg/ml concentration at the time of replicon transfection, and polio RNA replication was measured by luciferase assay. Expression of the GBF1 proteins was measured by Western blot; calnexin staining was used as a loading control (panel D, right).

    Article Snippet: Plasmid pCMV-Gluc used for the secretion rescue experiment was purchased from New England Biolabs.

    Techniques: Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Cell Viability Assay, Luciferase, Concentration Assay, Western Blot, Staining

    Sustained Gluc expression in vivo . Expression time course of Gluc from PB -based ( a,c ) or conventional ( b ) pDNA. 25 µg pIR-CMVGluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( a ). 25 µg pCMV-Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( b ). 25 µg pIR-EF1Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( c ). Blood samples were collected at the indicated time points, and Gluc activities in serum were measured. Each value represents mean ± SD ( n = 4–6). RLU, relative light unit.

    Journal: Molecular Therapy

    Article Title: piggyBac Transposon-mediated Long-term Gene Expression in Mice

    doi: 10.1038/mt.2009.302

    Figure Lengend Snippet: Sustained Gluc expression in vivo . Expression time course of Gluc from PB -based ( a,c ) or conventional ( b ) pDNA. 25 µg pIR-CMVGluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( a ). 25 µg pCMV-Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( b ). 25 µg pIR-EF1Gluc and 1 µg pFerH-PBTP (closed rhombuses), or pFerH-mcs (closed squares) were injected ( c ). Blood samples were collected at the indicated time points, and Gluc activities in serum were measured. Each value represents mean ± SD ( n = 4–6). RLU, relative light unit.

    Article Snippet: 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.

    Techniques: Expressing, In Vivo, Injection

    BFA-resistant GBF1 mutants M832L and A795E differ in their abilities to rescue protein secretion and cell viability in the presence of BFA. (A) HeLa cells were cotransfected with plasmid pCMV-Gluc expressing secreted Gaussia luciferase and either a control

    Journal: Journal of Virology

    Article Title: GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication ▿

    doi: 10.1128/JVI.01244-09

    Figure Lengend Snippet: BFA-resistant GBF1 mutants M832L and A795E differ in their abilities to rescue protein secretion and cell viability in the presence of BFA. (A) HeLa cells were cotransfected with plasmid pCMV-Gluc expressing secreted Gaussia luciferase and either a control

    Article Snippet: Plasmid pCMV-Gluc, which expresses secreted Gaussia luciferase, was purchased from New England Biolabs.

    Techniques: Plasmid Preparation, Expressing, Luciferase