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 "A survivin-driven, tumor-activatable minicircle system for prostate cancer theranostics"

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

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2021.01.007

    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
    Figure Legend 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

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

    3) 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

    4) 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

    5) Product Images from "Conformation and recognition of DNA modified by a new antitumor dinuclear PtII complex resistant to decomposition by sulfur nucleophiles"

    Article Title: Conformation and recognition of DNA modified by a new antitumor dinuclear PtII complex resistant to decomposition by sulfur nucleophiles

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2009.08.009

    Inhibition of RNA polymerase II transcription by DNA adducts of complex 1 or cisplatin. (A) Autoradiogram of the 8% PAA/8 M urea denaturing gel. Lanes: M, 100 bp DNA marker; 1, control, nonplatinated pCMV-Gluc substrate; 2, 3, and 4, pCMV-Gluc substrate
    Figure Legend Snippet: Inhibition of RNA polymerase II transcription by DNA adducts of complex 1 or cisplatin. (A) Autoradiogram of the 8% PAA/8 M urea denaturing gel. Lanes: M, 100 bp DNA marker; 1, control, nonplatinated pCMV-Gluc substrate; 2, 3, and 4, pCMV-Gluc substrate

    Techniques Used: Inhibition, Marker

    6) 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

    7) Product Images from "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 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

    Journal: Molecular pharmaceutics

    doi: 10.1021/mp200309x

    Inhibition of RNA polymerase II transcription by DNA adducts of complexes 1 , 2 and cisplatin. (A) Autoradiogram of the 8% PAA/8 M urea denaturing gel. Lanes: 1, unmodified substrate, no extract added; 2, control, unplatinated pCMV-Gluc substrate; 3, 4, 5 and 6, pCMV-Gluc substrate modified with cisplatin at r b = 5×10 −4 , 1×10 −3 , 2×10 −3 and 5×10 −3 , respectively; 7, 8, 9 and 10, pCMV-Gluc substrate modified by complex 1 at r b = 5×10 −4 , 1×10 −3 , 2×10 −3 and 5×10 −3 , respectively; 11, 12, 13 and 14, pCMV-Gluc substrate modified by complex 2 at r b = 5×10 −4 , 1×10 −3 , 2×10 −3 and 5×10 −3 , respectively. B. Quantitative assessment. The relative transcription was assed as follows: the amount of full length transcript at each r b was quantified (in % of total radioactivity in the lane) and calculated as the percentage of that generated by the control, undamaged template. Data represent results of two independent experiments and are expressed as mean percentages ±SEM. (■) cisplatin; (▲) 1 ; (●) 2 . C. Inhibition of RNA polymerase II transcription by the addition of increasing amount of exogenously platinated pUC19 DNA. The amount of full-length transcript in each lane is expressed as a mean fraction (±SEM) of that generated in the absence of exogenously added DNA (white bar). Black bars: transcription of undamaged DNA; gray bars: transcription of cisplatin modified DNA; light gray bars: transcription of DNA modified with 1; hatched bars: transcription of DNA modified with 2 .
    Figure Legend Snippet: Inhibition of RNA polymerase II transcription by DNA adducts of complexes 1 , 2 and cisplatin. (A) Autoradiogram of the 8% PAA/8 M urea denaturing gel. Lanes: 1, unmodified substrate, no extract added; 2, control, unplatinated pCMV-Gluc substrate; 3, 4, 5 and 6, pCMV-Gluc substrate modified with cisplatin at r b = 5×10 −4 , 1×10 −3 , 2×10 −3 and 5×10 −3 , respectively; 7, 8, 9 and 10, pCMV-Gluc substrate modified by complex 1 at r b = 5×10 −4 , 1×10 −3 , 2×10 −3 and 5×10 −3 , respectively; 11, 12, 13 and 14, pCMV-Gluc substrate modified by complex 2 at r b = 5×10 −4 , 1×10 −3 , 2×10 −3 and 5×10 −3 , respectively. B. Quantitative assessment. The relative transcription was assed as follows: the amount of full length transcript at each r b was quantified (in % of total radioactivity in the lane) and calculated as the percentage of that generated by the control, undamaged template. Data represent results of two independent experiments and are expressed as mean percentages ±SEM. (■) cisplatin; (▲) 1 ; (●) 2 . C. Inhibition of RNA polymerase II transcription by the addition of increasing amount of exogenously platinated pUC19 DNA. The amount of full-length transcript in each lane is expressed as a mean fraction (±SEM) of that generated in the absence of exogenously added DNA (white bar). Black bars: transcription of undamaged DNA; gray bars: transcription of cisplatin modified DNA; light gray bars: transcription of DNA modified with 1; hatched bars: transcription of DNA modified with 2 .

    Techniques Used: Inhibition, Modification, Radioactivity, Generated

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) Product Images from "A survivin-driven, tumor-activatable minicircle system for prostate cancer theranostics"

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

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2021.01.007

    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
    Figure Legend 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

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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

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    Plasmid Preparation:

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    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.
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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

    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

    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