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Promega β globin genes
Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and <t>β-globin</t> primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
β Globin Genes, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells"

Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

Journal: Scientific Reports

doi: 10.1038/srep05074

Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and β-globin primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
Figure Legend Snippet: Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and β-globin primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.

Techniques Used: Fractionation, Infection, Incubation, Real-time Polymerase Chain Reaction, Plasmid Preparation

LINE-1 transduction of the NP-2 cell line and its effect on VSV DNA formation. (a) LINE-1 transduction of the NP-2 cell line. The LINE-1 ORF1p and β-actin levels in the 293T, NP-2, NP-2/LINE-1 (L1.2), and NP-2/LINE-1 (L1.2 ORF2mut) cells were examined via Western blot using the anti-ORF1p IgY antibody. The ORF1p protein was observed at a molecular weight of ~41 kDa. The membrane was re-probed with an anti-β-actin antibody. The size marker at 38 kDa is indicated. Note that cropped western blots are shown and that full-length images are presented in the supplementary information . (b) The effect of LINE-1 transduction on VSV DNA production. The cells described above were infected with VSV, and the cell lysates were prepared. Real-time PCR was performed with either the N478-F/N681-R or β-globin primer pair. In addition, the VSV progeny produced by these cells were titrated and the VSV N mRNA expression levels were compared among these cells.
Figure Legend Snippet: LINE-1 transduction of the NP-2 cell line and its effect on VSV DNA formation. (a) LINE-1 transduction of the NP-2 cell line. The LINE-1 ORF1p and β-actin levels in the 293T, NP-2, NP-2/LINE-1 (L1.2), and NP-2/LINE-1 (L1.2 ORF2mut) cells were examined via Western blot using the anti-ORF1p IgY antibody. The ORF1p protein was observed at a molecular weight of ~41 kDa. The membrane was re-probed with an anti-β-actin antibody. The size marker at 38 kDa is indicated. Note that cropped western blots are shown and that full-length images are presented in the supplementary information . (b) The effect of LINE-1 transduction on VSV DNA production. The cells described above were infected with VSV, and the cell lysates were prepared. Real-time PCR was performed with either the N478-F/N681-R or β-globin primer pair. In addition, the VSV progeny produced by these cells were titrated and the VSV N mRNA expression levels were compared among these cells.

Techniques Used: Transduction, Western Blot, Molecular Weight, Marker, Infection, Real-time Polymerase Chain Reaction, Produced, Expressing

Cell-based differences in VSV DNA production. Various human cells were infected with VSV at an MOI of 0.1. The DNA was extracted 16 hr later and subjected to PCR amplification. Real-time PCR was performed using the N478-F/N681-R or β-globin primer pair. The copy numbers of the VSV N and β-globin genes in each cell line are shown in closed and open bars, respectively. The arrows indicate that the copy number per cell was below the detection limit. The positive cells are ordered according to the VSV N DNA level, followed by the VSV DNA-negative adherent cells. Subsequently, the VSV DNA-negative suspension cells are arranged in alphabetical order. The means ± SE of two independent experiments performed in duplicate are shown.
Figure Legend Snippet: Cell-based differences in VSV DNA production. Various human cells were infected with VSV at an MOI of 0.1. The DNA was extracted 16 hr later and subjected to PCR amplification. Real-time PCR was performed using the N478-F/N681-R or β-globin primer pair. The copy numbers of the VSV N and β-globin genes in each cell line are shown in closed and open bars, respectively. The arrows indicate that the copy number per cell was below the detection limit. The positive cells are ordered according to the VSV N DNA level, followed by the VSV DNA-negative adherent cells. Subsequently, the VSV DNA-negative suspension cells are arranged in alphabetical order. The means ± SE of two independent experiments performed in duplicate are shown.

Techniques Used: Infection, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

Related Articles

Polymerase Chain Reaction:

Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples
Article Snippet: .. PCR amplification as a functional assay of DNA quality The β-globin gene (HBB ) was amplified using GoTaq Colorless Master Mix (Promega), following the manufacturer's instructions. .. The reaction mixture of 0.2 μM for each of the β-globin gene primers (forward: 5′-CAACTTCATCCACGTTCACC-3′ and reverse: 5′-GAAGAGCCAAGGACAGGTAC-3′; IDT, Skokie, IL), 100 ng of DNA quantified by spectrophotometer (seven samples contained less than 100 ng), and GoTaq Colorless Master Mix.

Functional Assay:

Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples
Article Snippet: .. PCR amplification as a functional assay of DNA quality The β-globin gene (HBB ) was amplified using GoTaq Colorless Master Mix (Promega), following the manufacturer's instructions. .. The reaction mixture of 0.2 μM for each of the β-globin gene primers (forward: 5′-CAACTTCATCCACGTTCACC-3′ and reverse: 5′-GAAGAGCCAAGGACAGGTAC-3′; IDT, Skokie, IL), 100 ng of DNA quantified by spectrophotometer (seven samples contained less than 100 ng), and GoTaq Colorless Master Mix.

Amplification:

Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples
Article Snippet: .. PCR amplification as a functional assay of DNA quality The β-globin gene (HBB ) was amplified using GoTaq Colorless Master Mix (Promega), following the manufacturer's instructions. .. The reaction mixture of 0.2 μM for each of the β-globin gene primers (forward: 5′-CAACTTCATCCACGTTCACC-3′ and reverse: 5′-GAAGAGCCAAGGACAGGTAC-3′; IDT, Skokie, IL), 100 ng of DNA quantified by spectrophotometer (seven samples contained less than 100 ng), and GoTaq Colorless Master Mix.

Plasmid Preparation:

Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
Article Snippet: .. The copy number of each product was estimated using standard curves that were obtained using serial dilutions of the plasmids containing the VSV N and β-globin genes (pGEM®-T Easy vector) (Promega). .. PCR primers The oligonucleotide primers were synthesised by Hokkaido System Science Co., Ltd. (Sapporo, Japan).

Construct:

Article Title: Characterization of the human ?-globin downstream promoter region
Article Snippet: .. As templates in these experiments we used either the wild type or mutants of the β-globin gene in combination with the human growth hormone gene under control of the thymidine kinase promoter , or a CMV construct (Promega), which served as an internal control. ..

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    Promega β globin genes
    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and <t>β-globin</t> primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
    β Globin Genes, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β globin genes/product/Promega
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    β globin genes - by Bioz Stars, 2020-05
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    88
    Promega human β globin gene
    Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human <t>β-globin</t> intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.
    Human β Globin Gene, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human β globin gene/product/Promega
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    human β globin gene - by Bioz Stars, 2020-05
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    85
    Promega β globin utr luc rna
    Inhibition of <t>RNA</t> translation. (A) Transient replicon assay. A luciferase-reporting replicon (10 μg) was electroporated into BHK-21 cells. The transfected cells were immediately incubated with 0.9% DMSO or NITD-451 at the indicated concentrations. Luciferase activities were measured at 2, 4, 6, 16, 24, and 30 h posttransfection. (B) In vitro translation inhibition assay. A luciferase reporter replicon RNA of DENV-1 (strain Western Pacific) or reporter RNAs <t>(DENV-2–UTR-Luc</t> and <t>β-globin</t> UTR-Luc) were incubated with the human cell lysates (from the Pierce human in vitro protein expression kit) in the presence of NITD-451, NITD-452, cycloheximide, or DMSO (0.45%). After incubation at 30°C for 3.5 h, luciferase activity was measured by a luciferase assay system (Promega). Average results and standard deviations ( n = 3) are presented for all experiments.
    β Globin Utr Luc Rna, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β globin utr luc rna/product/Promega
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    β globin utr luc rna - by Bioz Stars, 2020-05
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    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and β-globin primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.

    Journal: Scientific Reports

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

    doi: 10.1038/srep05074

    Figure Lengend Snippet: Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and β-globin primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.

    Article Snippet: The copy number of each product was estimated using standard curves that were obtained using serial dilutions of the plasmids containing the VSV N and β-globin genes (pGEM®-T Easy vector) (Promega).

    Techniques: Fractionation, Infection, Incubation, Real-time Polymerase Chain Reaction, Plasmid Preparation

    LINE-1 transduction of the NP-2 cell line and its effect on VSV DNA formation. (a) LINE-1 transduction of the NP-2 cell line. The LINE-1 ORF1p and β-actin levels in the 293T, NP-2, NP-2/LINE-1 (L1.2), and NP-2/LINE-1 (L1.2 ORF2mut) cells were examined via Western blot using the anti-ORF1p IgY antibody. The ORF1p protein was observed at a molecular weight of ~41 kDa. The membrane was re-probed with an anti-β-actin antibody. The size marker at 38 kDa is indicated. Note that cropped western blots are shown and that full-length images are presented in the supplementary information . (b) The effect of LINE-1 transduction on VSV DNA production. The cells described above were infected with VSV, and the cell lysates were prepared. Real-time PCR was performed with either the N478-F/N681-R or β-globin primer pair. In addition, the VSV progeny produced by these cells were titrated and the VSV N mRNA expression levels were compared among these cells.

    Journal: Scientific Reports

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

    doi: 10.1038/srep05074

    Figure Lengend Snippet: LINE-1 transduction of the NP-2 cell line and its effect on VSV DNA formation. (a) LINE-1 transduction of the NP-2 cell line. The LINE-1 ORF1p and β-actin levels in the 293T, NP-2, NP-2/LINE-1 (L1.2), and NP-2/LINE-1 (L1.2 ORF2mut) cells were examined via Western blot using the anti-ORF1p IgY antibody. The ORF1p protein was observed at a molecular weight of ~41 kDa. The membrane was re-probed with an anti-β-actin antibody. The size marker at 38 kDa is indicated. Note that cropped western blots are shown and that full-length images are presented in the supplementary information . (b) The effect of LINE-1 transduction on VSV DNA production. The cells described above were infected with VSV, and the cell lysates were prepared. Real-time PCR was performed with either the N478-F/N681-R or β-globin primer pair. In addition, the VSV progeny produced by these cells were titrated and the VSV N mRNA expression levels were compared among these cells.

    Article Snippet: The copy number of each product was estimated using standard curves that were obtained using serial dilutions of the plasmids containing the VSV N and β-globin genes (pGEM®-T Easy vector) (Promega).

    Techniques: Transduction, Western Blot, Molecular Weight, Marker, Infection, Real-time Polymerase Chain Reaction, Produced, Expressing

    Cell-based differences in VSV DNA production. Various human cells were infected with VSV at an MOI of 0.1. The DNA was extracted 16 hr later and subjected to PCR amplification. Real-time PCR was performed using the N478-F/N681-R or β-globin primer pair. The copy numbers of the VSV N and β-globin genes in each cell line are shown in closed and open bars, respectively. The arrows indicate that the copy number per cell was below the detection limit. The positive cells are ordered according to the VSV N DNA level, followed by the VSV DNA-negative adherent cells. Subsequently, the VSV DNA-negative suspension cells are arranged in alphabetical order. The means ± SE of two independent experiments performed in duplicate are shown.

    Journal: Scientific Reports

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

    doi: 10.1038/srep05074

    Figure Lengend Snippet: Cell-based differences in VSV DNA production. Various human cells were infected with VSV at an MOI of 0.1. The DNA was extracted 16 hr later and subjected to PCR amplification. Real-time PCR was performed using the N478-F/N681-R or β-globin primer pair. The copy numbers of the VSV N and β-globin genes in each cell line are shown in closed and open bars, respectively. The arrows indicate that the copy number per cell was below the detection limit. The positive cells are ordered according to the VSV N DNA level, followed by the VSV DNA-negative adherent cells. Subsequently, the VSV DNA-negative suspension cells are arranged in alphabetical order. The means ± SE of two independent experiments performed in duplicate are shown.

    Article Snippet: The copy number of each product was estimated using standard curves that were obtained using serial dilutions of the plasmids containing the VSV N and β-globin genes (pGEM®-T Easy vector) (Promega).

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

    In vivo footprint analysis of a conserved E-box located in the murine adult β-globin downstream promoter region. NIH3T3L1 (lane 2) or MEL (lane 3) cells were treated with DMS. The DNA was purified from these cells and analyzed by linker ligation-mediated PCR as described in Materials and Methods. Lane 1 depicts the G sequencing ladder of the region. The position of the E-box at +60 is indicated on the right. The open circle on the left highlights a G residue that is consistently protected from cleavage by DMS in MEL cells.

    Journal: Nucleic Acids Research

    Article Title: Characterization of the human ?-globin downstream promoter region

    doi:

    Figure Lengend Snippet: In vivo footprint analysis of a conserved E-box located in the murine adult β-globin downstream promoter region. NIH3T3L1 (lane 2) or MEL (lane 3) cells were treated with DMS. The DNA was purified from these cells and analyzed by linker ligation-mediated PCR as described in Materials and Methods. Lane 1 depicts the G sequencing ladder of the region. The position of the E-box at +60 is indicated on the right. The open circle on the left highlights a G residue that is consistently protected from cleavage by DMS in MEL cells.

    Article Snippet: As templates in these experiments we used either the wild type or mutants of the β-globin gene in combination with the human growth hormone gene under control of the thymidine kinase promoter , or a CMV construct (Promega), which served as an internal control.

    Techniques: In Vivo, Purification, Ligation, Polymerase Chain Reaction, Sequencing

    Interaction of HLH proteins and NF-E2 with human β-globin downstream promoter sequences in vitro . ( A ) EMSA was carried out as described in Materials and Methods using a fragment encompassing the initiator/E-box as well as the E-box at +20 (WT). Radiolabeled DNA fragments were incubated with 8 µg MEL protein extracts for 45 min at 30°C (lanes 2–4 and 6–9; lanes 1 and 5, no protein). MEL protein extracts were pre-incubated either without (lane 2) or with a 50-fold excess (compared to the radiolabeled fragment) of unlabeled competitor DNA, either using the wild-type initiator (lane 3) or a mutant fragment in which the initiator/ E-box was mutated (INI mut , lane 4). Antibody supershift reactions were post-incubated either without (lane 6) or with specific antibodies raised against TFII-I (αTFII-I, lane 7), USF1 (αUSF1, lane 8) or USF2 (αUSF2, lane 9) for 20 min at 30°C. ( B ) Characterization of proteins interacting with an E-box motif located 60 bp downstream of the β-globin transcription initiation site. EMSA was carried out with a fragment encompassing the E-box element located 60 bp downstream of the start site of transcription. Radiolabeled fragment was incubated without (lane 1) or with 8 µg MEL protein extract (lanes 2–5). Proteins were post-incubated with specific antibodies raised against TFII-I (αTFII-I, lane 3), USF1 (αUSF1, lane 4) or USF2 (αUSF2, lane 5) for 20 min at 30°C. ( C ) Interaction of NF-E2 with a consensus MARE sequence from LCR element HS2. A radiolabeled oligonucleotide bearing the NF-E2-binding site from HS2 was incubated with 0.06 µg of a protein fraction enriched for a tethered p45/mafg protein (lane 2; lane 1 depicts the free probe). The binding reaction was pre-incubated with antibodies against p45 (lane 3) for 20 min at 30°C before addition of the radiolabeled fragment. ( D ) Interaction of NF-E2 with a MARE-sequence from the human β-globin downstream promoter region. A radiolabeled fragment bearing the β-globin downstream MARE like sequence was incubated with 0.2 µg of a protein fraction enriched for the tethered p45/mafg fusion protein (lane 2; lane 1 shows the free probe). Post-incubation with p45 antibodies (lane 3) was done as described in (B).

    Journal: Nucleic Acids Research

    Article Title: Characterization of the human ?-globin downstream promoter region

    doi:

    Figure Lengend Snippet: Interaction of HLH proteins and NF-E2 with human β-globin downstream promoter sequences in vitro . ( A ) EMSA was carried out as described in Materials and Methods using a fragment encompassing the initiator/E-box as well as the E-box at +20 (WT). Radiolabeled DNA fragments were incubated with 8 µg MEL protein extracts for 45 min at 30°C (lanes 2–4 and 6–9; lanes 1 and 5, no protein). MEL protein extracts were pre-incubated either without (lane 2) or with a 50-fold excess (compared to the radiolabeled fragment) of unlabeled competitor DNA, either using the wild-type initiator (lane 3) or a mutant fragment in which the initiator/ E-box was mutated (INI mut , lane 4). Antibody supershift reactions were post-incubated either without (lane 6) or with specific antibodies raised against TFII-I (αTFII-I, lane 7), USF1 (αUSF1, lane 8) or USF2 (αUSF2, lane 9) for 20 min at 30°C. ( B ) Characterization of proteins interacting with an E-box motif located 60 bp downstream of the β-globin transcription initiation site. EMSA was carried out with a fragment encompassing the E-box element located 60 bp downstream of the start site of transcription. Radiolabeled fragment was incubated without (lane 1) or with 8 µg MEL protein extract (lanes 2–5). Proteins were post-incubated with specific antibodies raised against TFII-I (αTFII-I, lane 3), USF1 (αUSF1, lane 4) or USF2 (αUSF2, lane 5) for 20 min at 30°C. ( C ) Interaction of NF-E2 with a consensus MARE sequence from LCR element HS2. A radiolabeled oligonucleotide bearing the NF-E2-binding site from HS2 was incubated with 0.06 µg of a protein fraction enriched for a tethered p45/mafg protein (lane 2; lane 1 depicts the free probe). The binding reaction was pre-incubated with antibodies against p45 (lane 3) for 20 min at 30°C before addition of the radiolabeled fragment. ( D ) Interaction of NF-E2 with a MARE-sequence from the human β-globin downstream promoter region. A radiolabeled fragment bearing the β-globin downstream MARE like sequence was incubated with 0.2 µg of a protein fraction enriched for the tethered p45/mafg fusion protein (lane 2; lane 1 shows the free probe). Post-incubation with p45 antibodies (lane 3) was done as described in (B).

    Article Snippet: As templates in these experiments we used either the wild type or mutants of the β-globin gene in combination with the human growth hormone gene under control of the thymidine kinase promoter , or a CMV construct (Promega), which served as an internal control.

    Techniques: In Vitro, Incubation, Mutagenesis, Sequencing, Binding Assay

    Characterization of protein–DNA interactions in the human β-globin downstream promoter region in vivo . ( A ) Chromatin immunoprecipitation (ChIP) experiment analyzing the interaction of proteins with the murine/human β-globin gene or the HS2 5′flanking region in MEL and K562 cells in vivo . Cells were incubated in 1% formaldehyde to crosslink protein–DNA and protein–protein interactions. After sonication, the cells were lysed and the chromatin was precipitated with either no antibody (lanes 3 and 11) or with antibodies specific for USF1 (lanes 4 and 12), USF2 (lanes 5 and 13), TFII-I (lanes 6 and 14), NF-E2 (p45, lanes 7 and 15) or acetylated histone H3 (lanes 8 and 16). DNA was purified from the precipitate and analyzed by PCR for the presence of the murine or human β-globin gene (210 and 321 bp, respectively, lanes 2–8) or the murine or human HS2 5′flank (336 and 565 bp, respectively, lanes 10–16). As positive controls, the input DNA was also analyzed by PCR (lanes 2 and 10). Lanes 1 and 9 show radiolabeled 100 bp markers. ( B ) Western blot analysis of protein extracts from MEL and K562 cells. Nuclear extracts were prepared from MEL or K562 cells and electrophoresed on denaturing polyacrylamide gels. Proteins were electroblotted to a nitrocellulose membrane, which was then hybridized to antibodies specific for USF1, USF2, TFII-I or NF-E2 (p45) as indicated. Bands were visualized by incubation with horseradish peroxidase-conjugated secondary antibody and autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Characterization of the human ?-globin downstream promoter region

    doi:

    Figure Lengend Snippet: Characterization of protein–DNA interactions in the human β-globin downstream promoter region in vivo . ( A ) Chromatin immunoprecipitation (ChIP) experiment analyzing the interaction of proteins with the murine/human β-globin gene or the HS2 5′flanking region in MEL and K562 cells in vivo . Cells were incubated in 1% formaldehyde to crosslink protein–DNA and protein–protein interactions. After sonication, the cells were lysed and the chromatin was precipitated with either no antibody (lanes 3 and 11) or with antibodies specific for USF1 (lanes 4 and 12), USF2 (lanes 5 and 13), TFII-I (lanes 6 and 14), NF-E2 (p45, lanes 7 and 15) or acetylated histone H3 (lanes 8 and 16). DNA was purified from the precipitate and analyzed by PCR for the presence of the murine or human β-globin gene (210 and 321 bp, respectively, lanes 2–8) or the murine or human HS2 5′flank (336 and 565 bp, respectively, lanes 10–16). As positive controls, the input DNA was also analyzed by PCR (lanes 2 and 10). Lanes 1 and 9 show radiolabeled 100 bp markers. ( B ) Western blot analysis of protein extracts from MEL and K562 cells. Nuclear extracts were prepared from MEL or K562 cells and electrophoresed on denaturing polyacrylamide gels. Proteins were electroblotted to a nitrocellulose membrane, which was then hybridized to antibodies specific for USF1, USF2, TFII-I or NF-E2 (p45) as indicated. Bands were visualized by incubation with horseradish peroxidase-conjugated secondary antibody and autoradiography.

    Article Snippet: As templates in these experiments we used either the wild type or mutants of the β-globin gene in combination with the human growth hormone gene under control of the thymidine kinase promoter , or a CMV construct (Promega), which served as an internal control.

    Techniques: In Vivo, Chromatin Immunoprecipitation, Incubation, Sonication, Purification, Polymerase Chain Reaction, Western Blot, Autoradiography

    ). Additional sequences required for the formation of active transcription complexes are MARE and E-box elements located in the downstream promoter region. These sequences are bound by NF-E2 (p45 and p18) and USF, respectively. It is proposed that in cells not expressing the β-globin gene (inactive), TFII-D is not bound and the initiator sequence is occupied by protein complexes consisting of TFII-I and USF2.

    Journal: Nucleic Acids Research

    Article Title: Characterization of the human ?-globin downstream promoter region

    doi:

    Figure Lengend Snippet: ). Additional sequences required for the formation of active transcription complexes are MARE and E-box elements located in the downstream promoter region. These sequences are bound by NF-E2 (p45 and p18) and USF, respectively. It is proposed that in cells not expressing the β-globin gene (inactive), TFII-D is not bound and the initiator sequence is occupied by protein complexes consisting of TFII-I and USF2.

    Article Snippet: As templates in these experiments we used either the wild type or mutants of the β-globin gene in combination with the human growth hormone gene under control of the thymidine kinase promoter , or a CMV construct (Promega), which served as an internal control.

    Techniques: Expressing, Sequencing

    Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human β-globin intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.

    Journal: Nucleic Acids Research

    Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2

    doi: 10.1093/nar/gkp497

    Figure Lengend Snippet: Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human β-globin intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.

    Article Snippet: For the pcDNAIntron-GlobinRen, the sequence corresponding to the intron of the human β-globin gene was amplified by PCR and cloned into the pcDNAGlobinRen vector previously digested by XbaI. pCI-mycORF57 contained the complete ORF57 coding sequence (first exon included) cloned in frame with the myc epitope, in pCI (Promega).

    Techniques: Luciferase, Construct, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Purification, Activity Assay, Quantitative RT-PCR

    Inhibition of RNA translation. (A) Transient replicon assay. A luciferase-reporting replicon (10 μg) was electroporated into BHK-21 cells. The transfected cells were immediately incubated with 0.9% DMSO or NITD-451 at the indicated concentrations. Luciferase activities were measured at 2, 4, 6, 16, 24, and 30 h posttransfection. (B) In vitro translation inhibition assay. A luciferase reporter replicon RNA of DENV-1 (strain Western Pacific) or reporter RNAs (DENV-2–UTR-Luc and β-globin UTR-Luc) were incubated with the human cell lysates (from the Pierce human in vitro protein expression kit) in the presence of NITD-451, NITD-452, cycloheximide, or DMSO (0.45%). After incubation at 30°C for 3.5 h, luciferase activity was measured by a luciferase assay system (Promega). Average results and standard deviations ( n = 3) are presented for all experiments.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Translation Inhibitor That Suppresses Dengue Virus In Vitro and In Vivo ▿ ▿ †

    doi: 10.1128/AAC.00620-11

    Figure Lengend Snippet: Inhibition of RNA translation. (A) Transient replicon assay. A luciferase-reporting replicon (10 μg) was electroporated into BHK-21 cells. The transfected cells were immediately incubated with 0.9% DMSO or NITD-451 at the indicated concentrations. Luciferase activities were measured at 2, 4, 6, 16, 24, and 30 h posttransfection. (B) In vitro translation inhibition assay. A luciferase reporter replicon RNA of DENV-1 (strain Western Pacific) or reporter RNAs (DENV-2–UTR-Luc and β-globin UTR-Luc) were incubated with the human cell lysates (from the Pierce human in vitro protein expression kit) in the presence of NITD-451, NITD-452, cycloheximide, or DMSO (0.45%). After incubation at 30°C for 3.5 h, luciferase activity was measured by a luciferase assay system (Promega). Average results and standard deviations ( n = 3) are presented for all experiments.

    Article Snippet: The cDNA plasmids for DENV-2–UTR-Luc RNA and β-globin UTR-Luc RNA were constructed by using overlapping PCR followed by cloning the PCR products into the plasmid vector pGL4.7 (Promega) at the NheI and BamHI sites and at the NheI and NruI sites, respectively.

    Techniques: Inhibition, Luciferase, Transfection, Incubation, In Vitro, Western Blot, Expressing, Activity Assay