Structured Review

Promega renilla luciferase genes
The RGG domain of Lsm4 is not required for mRNA decay or translational repression. (A) Northern blot assays showing the decay of an ARE-containing reporter mRNA (β-c-fos) in HeLa Tet-off cells treated with the siRNAs indicated and transiently expressing the Lsm4 proteins indicated. β-c-fos half-lives ( t 1/2 ) were calculated with the constitutively transcribed β-GAP internal control mRNA for normalization. Fold stabilization was calculated relative to the siLuc control condition in three experiments, and the standard errors of the means are indicated. (B) Northern blot assays showing the decay of endogenous H2A mRNA induced by treatment with 5 mM hydroxyurea in HEK 293 T-REx cells treated with the siRNAs indicated and stably expressing the Lsm4 proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA served as a normalization control. (C) Luciferase luminescence assays of cells transiently transfected with plasmids encoding the MS2 fusion proteins indicated, as well as a firefly luciferase reporter with six MS2 coat protein binding sites in its 3′ UTR (F-Luc-6xMS2) and a <t>Renilla</t> luciferase reporter (R-Luc) as an internal control. F-Luc-6xMS2 was normalized to R-Luc, and all samples were normalized to cells transfected with MS2 and the reporters alone. Error bars represent the standard errors of the means. **, P
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Images

1) Product Images from "The C-Terminal RGG Domain of Human Lsm4 Promotes Processing Body Formation Stimulated by Arginine Dimethylation"

Article Title: The C-Terminal RGG Domain of Human Lsm4 Promotes Processing Body Formation Stimulated by Arginine Dimethylation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01102-15

The RGG domain of Lsm4 is not required for mRNA decay or translational repression. (A) Northern blot assays showing the decay of an ARE-containing reporter mRNA (β-c-fos) in HeLa Tet-off cells treated with the siRNAs indicated and transiently expressing the Lsm4 proteins indicated. β-c-fos half-lives ( t 1/2 ) were calculated with the constitutively transcribed β-GAP internal control mRNA for normalization. Fold stabilization was calculated relative to the siLuc control condition in three experiments, and the standard errors of the means are indicated. (B) Northern blot assays showing the decay of endogenous H2A mRNA induced by treatment with 5 mM hydroxyurea in HEK 293 T-REx cells treated with the siRNAs indicated and stably expressing the Lsm4 proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA served as a normalization control. (C) Luciferase luminescence assays of cells transiently transfected with plasmids encoding the MS2 fusion proteins indicated, as well as a firefly luciferase reporter with six MS2 coat protein binding sites in its 3′ UTR (F-Luc-6xMS2) and a Renilla luciferase reporter (R-Luc) as an internal control. F-Luc-6xMS2 was normalized to R-Luc, and all samples were normalized to cells transfected with MS2 and the reporters alone. Error bars represent the standard errors of the means. **, P
Figure Legend Snippet: The RGG domain of Lsm4 is not required for mRNA decay or translational repression. (A) Northern blot assays showing the decay of an ARE-containing reporter mRNA (β-c-fos) in HeLa Tet-off cells treated with the siRNAs indicated and transiently expressing the Lsm4 proteins indicated. β-c-fos half-lives ( t 1/2 ) were calculated with the constitutively transcribed β-GAP internal control mRNA for normalization. Fold stabilization was calculated relative to the siLuc control condition in three experiments, and the standard errors of the means are indicated. (B) Northern blot assays showing the decay of endogenous H2A mRNA induced by treatment with 5 mM hydroxyurea in HEK 293 T-REx cells treated with the siRNAs indicated and stably expressing the Lsm4 proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA served as a normalization control. (C) Luciferase luminescence assays of cells transiently transfected with plasmids encoding the MS2 fusion proteins indicated, as well as a firefly luciferase reporter with six MS2 coat protein binding sites in its 3′ UTR (F-Luc-6xMS2) and a Renilla luciferase reporter (R-Luc) as an internal control. F-Luc-6xMS2 was normalized to R-Luc, and all samples were normalized to cells transfected with MS2 and the reporters alone. Error bars represent the standard errors of the means. **, P

Techniques Used: Northern Blot, Expressing, Stable Transfection, Luciferase, Transfection, Protein Binding

2) 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

3) Product Images from "The HoxC4 Homeodomain Protein Mediates Activation of the Immunoglobulin Heavy Chain 3′ hs1,2 Enhancer in Human B Cells"

Article Title: The HoxC4 Homeodomain Protein Mediates Activation of the Immunoglobulin Heavy Chain 3′ hs1,2 Enhancer in Human B Cells

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M407496200

The hs1,2 element is the strongest IgH 3′ enhancer Human 4B6 B cells were transfected with the indicated enhancer-promoter or promoter-only pGL3 luc -reporter gene vector to assess the enhancing activity of each C α 2 3′E H element. Three separate human promoters were used: V H 1 ( A ), ECS-I γ 3 ( B ), and β -globin ( C ). Luc activity is expressed as relative light units. Data are the mean values of three independent experiments plus standard deviation.
Figure Legend Snippet: The hs1,2 element is the strongest IgH 3′ enhancer Human 4B6 B cells were transfected with the indicated enhancer-promoter or promoter-only pGL3 luc -reporter gene vector to assess the enhancing activity of each C α 2 3′E H element. Three separate human promoters were used: V H 1 ( A ), ECS-I γ 3 ( B ), and β -globin ( C ). Luc activity is expressed as relative light units. Data are the mean values of three independent experiments plus standard deviation.

Techniques Used: Transfection, Plasmid Preparation, Activity Assay, Standard Deviation

Regions 1, 2, and 3 are necessary but individually are not sufficient for transcription enhancement Human 4B6 B cells were transfected with the indicated pGL3 luc -reporter gene vectors to measure the contribution of different DNA regions to the overall hs1,2-enhancing activity. Sequential 5′- and 3′-end truncation, internal deletion, and subfragment mutants of the hs1,2 sequence were created and subcloned into the pGL3 vector driven by V H 1 or ECS-I γ 3 promoter. The basal enhancing activity was determined and expressed as relative light units. Data are the mean values of three independent experiments plus standard deviations.
Figure Legend Snippet: Regions 1, 2, and 3 are necessary but individually are not sufficient for transcription enhancement Human 4B6 B cells were transfected with the indicated pGL3 luc -reporter gene vectors to measure the contribution of different DNA regions to the overall hs1,2-enhancing activity. Sequential 5′- and 3′-end truncation, internal deletion, and subfragment mutants of the hs1,2 sequence were created and subcloned into the pGL3 vector driven by V H 1 or ECS-I γ 3 promoter. The basal enhancing activity was determined and expressed as relative light units. Data are the mean values of three independent experiments plus standard deviations.

Techniques Used: Transfection, Activity Assay, Sequencing, Plasmid Preparation

The HoxC4 and HoxC4/Oct cis -elements are required for full hs1,2-enhancing activity The hs1,2 HoxC4 and HoxC4/Oct-binding sites were mutagenized by site-directed PCR, resulting in single and double mutant enhancer pGL3 luc -reporter gene constructs driven by the V H 1, ECS-I γ 3, or β -globin promoter. Each construct was used to transfect 4B6 cells to measure Luc activity. Data are represented as percentage of wild-type hs1,2 (regions 1–3)-enhancing activity and are the mean values of three independent experiments plus standard deviations.
Figure Legend Snippet: The HoxC4 and HoxC4/Oct cis -elements are required for full hs1,2-enhancing activity The hs1,2 HoxC4 and HoxC4/Oct-binding sites were mutagenized by site-directed PCR, resulting in single and double mutant enhancer pGL3 luc -reporter gene constructs driven by the V H 1, ECS-I γ 3, or β -globin promoter. Each construct was used to transfect 4B6 cells to measure Luc activity. Data are represented as percentage of wild-type hs1,2 (regions 1–3)-enhancing activity and are the mean values of three independent experiments plus standard deviations.

Techniques Used: Activity Assay, Binding Assay, Polymerase Chain Reaction, Mutagenesis, Construct

4) Product Images from "Characterization of the human ?-globin downstream promoter region"

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

Journal: Nucleic Acids Research

doi:

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.
Figure Legend 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.

Techniques Used: 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).
Figure Legend 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).

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Expressing, Sequencing

5) Product Images from "Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2"

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp497

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.
Figure Legend 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.

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

6) Product Images from "MPN patients harbor recurrent truncating mutations in transcription factor NF-E2"

Article Title: MPN patients harbor recurrent truncating mutations in transcription factor NF-E2

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20120521

Transactivating activity of mutant NF-E2 proteins and their effect on WT NF-E2 activity. Plasmids encoding a β-globin promoter-luciferase construct (A; Igarashi et al., 1994 ) or a reporter construct encoding 5.2 kb of the β1-tubulin promoter coupled to the luciferase reporter gene (B) were cotransfected into HEK-293 cells either with expression vectors for MafG (white bars), WT NF-E2 (black bars), or various NF-E2 mutants (gray bars) as indicated. Luciferase activity was measured 16 h after transfection and normalized for transfection efficiency by determination of Renilla luciferase activity from a cotransfected vector. Activity of MafG alone was set at 1 and fold activity relative to this control is depicted. Bar graphs represent the mean ± SEM of four independent experiments, each performed in duplicate. Statistical analysis was done by one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. *, P
Figure Legend Snippet: Transactivating activity of mutant NF-E2 proteins and their effect on WT NF-E2 activity. Plasmids encoding a β-globin promoter-luciferase construct (A; Igarashi et al., 1994 ) or a reporter construct encoding 5.2 kb of the β1-tubulin promoter coupled to the luciferase reporter gene (B) were cotransfected into HEK-293 cells either with expression vectors for MafG (white bars), WT NF-E2 (black bars), or various NF-E2 mutants (gray bars) as indicated. Luciferase activity was measured 16 h after transfection and normalized for transfection efficiency by determination of Renilla luciferase activity from a cotransfected vector. Activity of MafG alone was set at 1 and fold activity relative to this control is depicted. Bar graphs represent the mean ± SEM of four independent experiments, each performed in duplicate. Statistical analysis was done by one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. *, P

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

7) Product Images from "Regulation of I?B? expression involves both NF-?B and the MAP kinase signaling pathways"

Article Title: Regulation of I?B? expression involves both NF-?B and the MAP kinase signaling pathways

Journal: Journal of Inflammation (London, England)

doi: 10.1186/1476-9255-2-10

Ex vivo measurement of the effect of SB203580 on LPS-induced luciferase expression. A. Selected organs were harvested from SB203580 pre-treated mice and LPS treated control mice at 4 hours after the LPS injection. * indicates a significant difference between vehicle (DMSO) + LPS and SB203580 + LPS (p = 0.05; sign test). B. Northern blot analysis of IκBα mRNA in the liver tissue. I κ B α- luc transgenic mice were sacrificed at 3 hours after LPS injection. Liver tissue was harvested and processed for RNA isolation. A total of 2 μg of RNA was analyzed by Northern blot. Equal loading was demonstrated by 28S rRNA.
Figure Legend Snippet: Ex vivo measurement of the effect of SB203580 on LPS-induced luciferase expression. A. Selected organs were harvested from SB203580 pre-treated mice and LPS treated control mice at 4 hours after the LPS injection. * indicates a significant difference between vehicle (DMSO) + LPS and SB203580 + LPS (p = 0.05; sign test). B. Northern blot analysis of IκBα mRNA in the liver tissue. I κ B α- luc transgenic mice were sacrificed at 3 hours after LPS injection. Liver tissue was harvested and processed for RNA isolation. A total of 2 μg of RNA was analyzed by Northern blot. Equal loading was demonstrated by 28S rRNA.

Techniques Used: Ex Vivo, Luciferase, Expressing, Mouse Assay, Injection, Northern Blot, Transgenic Assay, Isolation

Imaging analysis of luciferase expression in I κ B α- luc transgenic mice treated with LPS. A. I κ B α- luc transgenic mice were imaged at T = 0, 2, 4, 7 and 24 hours after treatment with LPS (1 mg/kg, i.p ., n = 4 for males, n = 6 for females). Representative mice from each treatment group are shown. The color overlay on the image represents the photons/second emitted from the mouse body in accord with the pseudo-color scale shown on the right of the images. Red represents the highest photons/sec while blue represents the lowest photons/sec. B. Quantification of the luciferase signal from the abdominal region of the body. Data are means luciferase activity (billion photon/second) ± SE. Statistical analysis was done for male and female combined data. * indicates a significant induction of luciferase signal by LPS (P = 0.002). C. Northern blot analysis of IκBα mRNA in the liver tissue. Liver tissue was harvested from saline (control) or LPS treated I κ B α- luc female mice at 4 hours after treatment and processed for RNA isolation. A total of 2 μg of RNA was analyzed by Northern blot. Equal loading was demonstrated by 28S rRNA.
Figure Legend Snippet: Imaging analysis of luciferase expression in I κ B α- luc transgenic mice treated with LPS. A. I κ B α- luc transgenic mice were imaged at T = 0, 2, 4, 7 and 24 hours after treatment with LPS (1 mg/kg, i.p ., n = 4 for males, n = 6 for females). Representative mice from each treatment group are shown. The color overlay on the image represents the photons/second emitted from the mouse body in accord with the pseudo-color scale shown on the right of the images. Red represents the highest photons/sec while blue represents the lowest photons/sec. B. Quantification of the luciferase signal from the abdominal region of the body. Data are means luciferase activity (billion photon/second) ± SE. Statistical analysis was done for male and female combined data. * indicates a significant induction of luciferase signal by LPS (P = 0.002). C. Northern blot analysis of IκBα mRNA in the liver tissue. Liver tissue was harvested from saline (control) or LPS treated I κ B α- luc female mice at 4 hours after treatment and processed for RNA isolation. A total of 2 μg of RNA was analyzed by Northern blot. Equal loading was demonstrated by 28S rRNA.

Techniques Used: Imaging, Luciferase, Expressing, Transgenic Assay, Mouse Assay, Size-exclusion Chromatography, Activity Assay, Northern Blot, Isolation

Effect of bortezomib pre-treatment on the LPS-induced luciferase activity in selected tissues in I κ B α- luc male ( A ) and female ( B ) mice (n = 3 for both genders). Mice were injected with bortezomib (1 mg/kg, i.v .) 1 hour prior to the LPS treatment (1 mg/kg, i.p .). Mice treated with LPS alone were used as positive controls. Organs were harvested from all the mice at 3 hours after the LPS injection and processed for luciferase activity.* indicates a significant reduction in signal by bortezomib (P = 0.05). C. Northern blot analysis of IκBα mRNA in the liver tissue. I κ B α- luc transgenic mice were sacrificed at 3 hours after LPS injection. Liver tissue was harvested and processed for RNA isolation. A total of 2 μg of RNA was analyzed by Northern blot. Equal loading was demonstrated by 28S rRNA.
Figure Legend Snippet: Effect of bortezomib pre-treatment on the LPS-induced luciferase activity in selected tissues in I κ B α- luc male ( A ) and female ( B ) mice (n = 3 for both genders). Mice were injected with bortezomib (1 mg/kg, i.v .) 1 hour prior to the LPS treatment (1 mg/kg, i.p .). Mice treated with LPS alone were used as positive controls. Organs were harvested from all the mice at 3 hours after the LPS injection and processed for luciferase activity.* indicates a significant reduction in signal by bortezomib (P = 0.05). C. Northern blot analysis of IκBα mRNA in the liver tissue. I κ B α- luc transgenic mice were sacrificed at 3 hours after LPS injection. Liver tissue was harvested and processed for RNA isolation. A total of 2 μg of RNA was analyzed by Northern blot. Equal loading was demonstrated by 28S rRNA.

Techniques Used: Luciferase, Activity Assay, Mouse Assay, Injection, Northern Blot, Transgenic Assay, Isolation

8) Product Images from "Severe Acute Respiratory Syndrome Coronavirus Protein nsp1 Is a Novel Eukaryotic Translation Inhibitor That Represses Multiple Steps of Translation Initiation"

Article Title: Severe Acute Respiratory Syndrome Coronavirus Protein nsp1 Is a Novel Eukaryotic Translation Inhibitor That Represses Multiple Steps of Translation Initiation

Journal: Journal of Virology

doi: 10.1128/JVI.01958-12

nsp1-CD lacks the endonucleolytic RNA cleavage function. (A) GLA RNA was incubated in RRL in the presence of GST, nsp1, nsp1-mt, or nsp1-CD at 30°C for 20 min. (Left) After incubation, a small aliquot was removed and used to determine rLuc activities.
Figure Legend Snippet: nsp1-CD lacks the endonucleolytic RNA cleavage function. (A) GLA RNA was incubated in RRL in the presence of GST, nsp1, nsp1-mt, or nsp1-CD at 30°C for 20 min. (Left) After incubation, a small aliquot was removed and used to determine rLuc activities.

Techniques Used: Incubation

nsp1-CD lacks endonucleolytic RNA cleavage activity. (A) Schematic diagram of the structures of full-length dicistronic Ren-EMCV-FF RNA transcripts (full length), RNA 1, containing the 5′ rLuc gene and intercistronic IRES sequence, and RNA 2,
Figure Legend Snippet: nsp1-CD lacks endonucleolytic RNA cleavage activity. (A) Schematic diagram of the structures of full-length dicistronic Ren-EMCV-FF RNA transcripts (full length), RNA 1, containing the 5′ rLuc gene and intercistronic IRES sequence, and RNA 2,

Techniques Used: Activity Assay, Sequencing

9) 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

Sequence analysis of the 5′ and 3′ ends of the VSV DNA. (a) Schematic diagram of the inverse PCR method used to amplify the 5′ and 3′ ends of the VSV DNA. According to the results shown in Fig. 3 , we proposed that the VSV mRNA was reverse transcribed to DNA. The start (nucleotide 51) and end (nucleotide 1376) sites of the VSV N mRNA are indicated (GenBank: J02428.1). The cell lysates were treated with RNase H to digest the VSV RNA and then incubated with T4 RNA ligase to induce intra-molecular DNA end joining. The DNA was then amplified across the junction via nested PCR and sequenced. (b) The VSV DNA sequences of the head-to-tail junctions are shown. (A) VSV DNAs from four clones of 293T cells, three clones of JHH-7 cells, and one clone of Hep G2 cells are shown. All clones contained sequences corresponding to the 5′ end of the N mRNA (nt position 1376), followed by polyA tails consisting of 11 to 78 adenylate residues. (B) No additional G residue was found in two clones from the NP-2/LINE-1 (L1.2) cells infected with VSV.
Figure Legend Snippet: Sequence analysis of the 5′ and 3′ ends of the VSV DNA. (a) Schematic diagram of the inverse PCR method used to amplify the 5′ and 3′ ends of the VSV DNA. According to the results shown in Fig. 3 , we proposed that the VSV mRNA was reverse transcribed to DNA. The start (nucleotide 51) and end (nucleotide 1376) sites of the VSV N mRNA are indicated (GenBank: J02428.1). The cell lysates were treated with RNase H to digest the VSV RNA and then incubated with T4 RNA ligase to induce intra-molecular DNA end joining. The DNA was then amplified across the junction via nested PCR and sequenced. (b) The VSV DNA sequences of the head-to-tail junctions are shown. (A) VSV DNAs from four clones of 293T cells, three clones of JHH-7 cells, and one clone of Hep G2 cells are shown. All clones contained sequences corresponding to the 5′ end of the N mRNA (nt position 1376), followed by polyA tails consisting of 11 to 78 adenylate residues. (B) No additional G residue was found in two clones from the NP-2/LINE-1 (L1.2) cells infected with VSV.

Techniques Used: Sequencing, Inverse PCR, Incubation, Amplification, Nested PCR, Clone Assay, Infection

Detection of VSV DNA in 293T human cells. (a) DNA was extracted from 293T cells at 0, 1, 2, 4, 6, or 16 hr after infection with VSV at an MOI of 0.1 and subjected to PCR amplification for the VSV N gene using the N478-F/N681-R primer pair. The VSV inoculum (Sup) (lane 7) and mock-infected samples (lane 8) were included as controls. The samples prepared 1 hr after inoculation (lane 2) or VSV Sup did not produce VSV DNA. The DNA sequence of the PCR product obtained from the cells harvested at 16 hr post-infection (lane 6) was verified to be the expected VSV N amplicon. (b) DNA samples were harvested at 16 hr post-infection and were treated with RNase A or DNase I and subsequently used for PCR. The VSV DNA was not detected after DNase I treatment (lane 2). All samples used in Figures 1a and 1b were prepared in a single experiment, and cropped gel images are shown.
Figure Legend Snippet: Detection of VSV DNA in 293T human cells. (a) DNA was extracted from 293T cells at 0, 1, 2, 4, 6, or 16 hr after infection with VSV at an MOI of 0.1 and subjected to PCR amplification for the VSV N gene using the N478-F/N681-R primer pair. The VSV inoculum (Sup) (lane 7) and mock-infected samples (lane 8) were included as controls. The samples prepared 1 hr after inoculation (lane 2) or VSV Sup did not produce VSV DNA. The DNA sequence of the PCR product obtained from the cells harvested at 16 hr post-infection (lane 6) was verified to be the expected VSV N amplicon. (b) DNA samples were harvested at 16 hr post-infection and were treated with RNase A or DNase I and subsequently used for PCR. The VSV DNA was not detected after DNase I treatment (lane 2). All samples used in Figures 1a and 1b were prepared in a single experiment, and cropped gel images are shown.

Techniques Used: Infection, Polymerase Chain Reaction, Amplification, Sequencing

Effects of reverse transcriptase inhibitors on VSV DNA production. The synthesis of VSV DNA in the 293T and NP-2/LINE-1 (L1.2) cells in the presence of AZT, ddI, or ddT was examined via real-time PCR using the VSV N primers. We also tested the effects of these drugs on infection with recombinant MuLV pseudotype virus or HIV-1 strain IIIB. The means ± SE of two independent experiments performed in duplicate are shown.
Figure Legend Snippet: Effects of reverse transcriptase inhibitors on VSV DNA production. The synthesis of VSV DNA in the 293T and NP-2/LINE-1 (L1.2) cells in the presence of AZT, ddI, or ddT was examined via real-time PCR using the VSV N primers. We also tested the effects of these drugs on infection with recombinant MuLV pseudotype virus or HIV-1 strain IIIB. The means ± SE of two independent experiments performed in duplicate are shown.

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

10) Product Images from "Cellular and Viral Factors Regulate the Varicella-Zoster Virus gE Promoter during Viral Replication ▿"

Article Title: Cellular and Viral Factors Regulate the Varicella-Zoster Virus gE Promoter during Viral Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00553-07

Elements of the gE promoter required for VZV transactivator activity. Melanoma cells were transfected with the gE promoter construct and the plasmids expressing the VZV transactivators IE62, ORF61, IE63, and IE4, either individually or in combination with IE62 (A) and the ΔI, ΔII, and ΔIII gE promoter deletion constructs and pCMV-IE62 (B). Cells were harvested 24 h after transfection, and the luciferase assay was performed. Transfections were normalized with the protein lysate concentration (indicated by the symbol “[ ]”) determined by Bradford assay (Bio-Rad) (A) or by cotransfecting the phRL plasmid without any promoter driving the Renilla gene (B). The bars indicate the means ± the standard deviations of three independent transfections made in duplicate.
Figure Legend Snippet: Elements of the gE promoter required for VZV transactivator activity. Melanoma cells were transfected with the gE promoter construct and the plasmids expressing the VZV transactivators IE62, ORF61, IE63, and IE4, either individually or in combination with IE62 (A) and the ΔI, ΔII, and ΔIII gE promoter deletion constructs and pCMV-IE62 (B). Cells were harvested 24 h after transfection, and the luciferase assay was performed. Transfections were normalized with the protein lysate concentration (indicated by the symbol “[ ]”) determined by Bradford assay (Bio-Rad) (A) or by cotransfecting the phRL plasmid without any promoter driving the Renilla gene (B). The bars indicate the means ± the standard deviations of three independent transfections made in duplicate.

Techniques Used: Activity Assay, Transfection, Construct, Expressing, Luciferase, Concentration Assay, Bradford Assay, Plasmid Preparation

Effect of Sp1 mutations on transactivation activity of the gE promoter by IE62. (A) Schematic representation of the four probes used in EMSA. wt, wild-type sequence; SP1A, mutation of the A site; SP1B, mutation of the B site; SP1AB, mutation of the A and B sites. (B) EMSA results. Sp1 binds specifically to the gE promoter, as unlabeled wild-type probe competes for binding. Mutation of the Sp1 binding sites abolished complex formation. (C) Luciferase assay in melanoma cells at 24 h after transfection with the Sp1 mutant constructs and pCMV-IE62 vector. Transfections were normalized by cotransfecting the phRL plasmid without any promoter driving the Renilla gene. The results shown were obtained in three independent transfections made in duplicate. The standard deviation is indicated.
Figure Legend Snippet: Effect of Sp1 mutations on transactivation activity of the gE promoter by IE62. (A) Schematic representation of the four probes used in EMSA. wt, wild-type sequence; SP1A, mutation of the A site; SP1B, mutation of the B site; SP1AB, mutation of the A and B sites. (B) EMSA results. Sp1 binds specifically to the gE promoter, as unlabeled wild-type probe competes for binding. Mutation of the Sp1 binding sites abolished complex formation. (C) Luciferase assay in melanoma cells at 24 h after transfection with the Sp1 mutant constructs and pCMV-IE62 vector. Transfections were normalized by cotransfecting the phRL plasmid without any promoter driving the Renilla gene. The results shown were obtained in three independent transfections made in duplicate. The standard deviation is indicated.

Techniques Used: Activity Assay, Sequencing, Mutagenesis, Binding Assay, Luciferase, Transfection, Construct, Plasmid Preparation, Standard Deviation

11) Product Images from "Specific repression of ?-globin promoter activity by nuclear ferritin"

Article Title: Specific repression of ?-globin promoter activity by nuclear ferritin

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.151147098

Binding of ferritin chains from human heart and liver to the distal promoter of the human β-globin gene. The Rsa fragment of the β-globin promoter (−222/−128) was end-labeled with 32 ), with 1 μl of K562 nuclear extract (lane 2) or 2–4 ng of purified proteins, as follows: 2 ng of human liver ferritin (lane 3), 4 ng of human heart ferritin (lane 4), 2 ng each of human transferrin (lane 5) and apotransferrin (lane 6). Shift bands are denoted by arrowheads at the left. Lane 1 contained only DNA. A second fragment of the β-globin promoter (−127/+20) gave no shift bands with either 2 ng (lane 7) or 4 ng (lane 8) of liver ferritin or 4 ng of heart ferritin (lane 9). Only ferritin enriched in H-chains gave a strong shift (lane 4). The DNA-binding reactions for this figure were carried out at 37°C.
Figure Legend Snippet: Binding of ferritin chains from human heart and liver to the distal promoter of the human β-globin gene. The Rsa fragment of the β-globin promoter (−222/−128) was end-labeled with 32 ), with 1 μl of K562 nuclear extract (lane 2) or 2–4 ng of purified proteins, as follows: 2 ng of human liver ferritin (lane 3), 4 ng of human heart ferritin (lane 4), 2 ng each of human transferrin (lane 5) and apotransferrin (lane 6). Shift bands are denoted by arrowheads at the left. Lane 1 contained only DNA. A second fragment of the β-globin promoter (−127/+20) gave no shift bands with either 2 ng (lane 7) or 4 ng (lane 8) of liver ferritin or 4 ng of heart ferritin (lane 9). Only ferritin enriched in H-chains gave a strong shift (lane 4). The DNA-binding reactions for this figure were carried out at 37°C.

Techniques Used: Binding Assay, Labeling, Purification

Cotransfection experiments demonstrating ferritin-H repression of the β-globin promoter and loss of ability to repress when the ferritin binding site (CAGTGC) is mutated. Transfections of CV-1 cells were performed with a constant amount (6 μg) of total plasmid DNAs mixed with 8 μl of DMRIE-C added to 2 × 10 6 CV-1 cells, such that each transfection had 2 μg of β-CAT plasmid (W = wt, or M = mutant), ±1 μg of EKLF, ±3 μg of F H (ferritin-H expression plasmid), with the difference made up to 6 μg with pEGFP. Reporter gene activity, expressed as ng of CAT per mg of cellular protein (measured by ELISA), is shown for the following combinations with either native (W) or mutant (M) β-CAT plasmids: the nonstimulated human β-globin promoter (open bars); the β-globin promoter stimulated by a cotransfected EKLF effector plasmid (hatched bars); and EKLF-stimulated β-globin promoter cotransfected with a ferritin-H expression plasmid (solid bars). ( n = 3 transfections per data set; bars = SEM). Construction of reporter plasmids (diagrammed above the histogram) is described in Materials and Methods .
Figure Legend Snippet: Cotransfection experiments demonstrating ferritin-H repression of the β-globin promoter and loss of ability to repress when the ferritin binding site (CAGTGC) is mutated. Transfections of CV-1 cells were performed with a constant amount (6 μg) of total plasmid DNAs mixed with 8 μl of DMRIE-C added to 2 × 10 6 CV-1 cells, such that each transfection had 2 μg of β-CAT plasmid (W = wt, or M = mutant), ±1 μg of EKLF, ±3 μg of F H (ferritin-H expression plasmid), with the difference made up to 6 μg with pEGFP. Reporter gene activity, expressed as ng of CAT per mg of cellular protein (measured by ELISA), is shown for the following combinations with either native (W) or mutant (M) β-CAT plasmids: the nonstimulated human β-globin promoter (open bars); the β-globin promoter stimulated by a cotransfected EKLF effector plasmid (hatched bars); and EKLF-stimulated β-globin promoter cotransfected with a ferritin-H expression plasmid (solid bars). ( n = 3 transfections per data set; bars = SEM). Construction of reporter plasmids (diagrammed above the histogram) is described in Materials and Methods .

Techniques Used: Cotransfection, Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Nuclear anti-ferritin-reactive protein binds the β-globin promoter. ( a ) Diagram of the human β-globin locus, a 5′ region of the β-globin gene (−250 to +20), and specific segments used in b , i.e., the distal promoter (−222/−128) and two double-stranded oligonucleotides, −164/−128 and −232/−188. ( b ) Binding of a ferritin-family protein from K562 cell nuclear extracts to the −222/−128 β-globin region, and localization of the binding to the −164/−128 segment by using an antibody supershift assay described under Materials and Methods .
Figure Legend Snippet: Nuclear anti-ferritin-reactive protein binds the β-globin promoter. ( a ) Diagram of the human β-globin locus, a 5′ region of the β-globin gene (−250 to +20), and specific segments used in b , i.e., the distal promoter (−222/−128) and two double-stranded oligonucleotides, −164/−128 and −232/−188. ( b ) Binding of a ferritin-family protein from K562 cell nuclear extracts to the −222/−128 β-globin region, and localization of the binding to the −164/−128 segment by using an antibody supershift assay described under Materials and Methods .

Techniques Used: Binding Assay

12) Product Images from "Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples"

Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples

Journal: Biopreservation and Biobanking

doi: 10.1089/bio.2018.0148

Amplification of β-globin gene region from DNA isolated from LBC samples. Lane L: DNA ladder; Lane 1: negative control; Lanes 2–18: amplicon DNA of LBC samples; Lane 19: positive control amplicon DNAg previously known; Conditions: 8 μL of amplicon DNA in agarose gel at 2% at 95 volts during 90 minutes.
Figure Legend Snippet: Amplification of β-globin gene region from DNA isolated from LBC samples. Lane L: DNA ladder; Lane 1: negative control; Lanes 2–18: amplicon DNA of LBC samples; Lane 19: positive control amplicon DNAg previously known; Conditions: 8 μL of amplicon DNA in agarose gel at 2% at 95 volts during 90 minutes.

Techniques Used: Amplification, Isolation, Negative Control, Positive Control, Agarose Gel Electrophoresis

13) Product Images from "Mechanism of escape from nonsense-mediated mRNA decay of human ?-globin transcripts with nonsense mutations in the first exon"

Article Title: Mechanism of escape from nonsense-mediated mRNA decay of human ?-globin transcripts with nonsense mutations in the first exon

Journal: RNA

doi: 10.1261/rna.2401811

β-globin exon 1 is bisected by a sharp border between nonsense codons that do or do not activate NMD. Northern blot analysis of HeLa cells transiently transfected with normal (N) or nonsense mutated β-globin genes with the endogenous 5′
Figure Legend Snippet: β-globin exon 1 is bisected by a sharp border between nonsense codons that do or do not activate NMD. Northern blot analysis of HeLa cells transiently transfected with normal (N) or nonsense mutated β-globin genes with the endogenous 5′

Techniques Used: Northern Blot, Transfection

Translation reinitiation after termination codons in exon 1. ( A ) Schematic representation of the β-globinv gene, which contains a venus open reading frame (ORF) fused in-frame to the 3′ end of the β-globin sequence. (Bars) The
Figure Legend Snippet: Translation reinitiation after termination codons in exon 1. ( A ) Schematic representation of the β-globinv gene, which contains a venus open reading frame (ORF) fused in-frame to the 3′ end of the β-globin sequence. (Bars) The

Techniques Used: Sequencing

Read-through does not account for NMD insensitivity in exon 1. ( A ) Schematic representation of the β-globin gene with nonsense mutations at codons 16 and 39. (Shaded) NMD-resistant 5′ region in the β-globin mRNA. ( B ) Northern blot
Figure Legend Snippet: Read-through does not account for NMD insensitivity in exon 1. ( A ) Schematic representation of the β-globin gene with nonsense mutations at codons 16 and 39. (Shaded) NMD-resistant 5′ region in the β-globin mRNA. ( B ) Northern blot

Techniques Used: Northern Blot

Mutation of methionine 55 restores NMD sensitivity of nonsense mutations in exon 1. ( A ) Schematic representation of the β-globin gene with nonsense mutations at positions 3, 9, 16, 26, and 39. (Shaded) NMD-resistant 5′ region. Met55 designates
Figure Legend Snippet: Mutation of methionine 55 restores NMD sensitivity of nonsense mutations in exon 1. ( A ) Schematic representation of the β-globin gene with nonsense mutations at positions 3, 9, 16, 26, and 39. (Shaded) NMD-resistant 5′ region. Met55 designates

Techniques Used: Mutagenesis

14) Product Images from "A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression"

Article Title: A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression

Journal: Skeletal Muscle

doi: 10.1186/s13395-018-0181-y

A schematic of the HSA-rtTA transgene. The promoter and first exon (− 2,000 to + 239 relative to the transcription start site) of the human skeletal muscle α-actin (HSA) gene regulates expression of an optimized reverse tetracycline transactivator (rtTA) gene which has been reported to be sevenfold more active and 100-fold more doxycycline sensitive than the original Tet-On system [ 8 ]. The β-globin intron ΙΙ (BGI) and poly(A) tail were incorporated into the transgene to ensure proper splicing and transcript stability, respectively. The positions of the PCR primers used for genotyping are indicated by half-arrows
Figure Legend Snippet: A schematic of the HSA-rtTA transgene. The promoter and first exon (− 2,000 to + 239 relative to the transcription start site) of the human skeletal muscle α-actin (HSA) gene regulates expression of an optimized reverse tetracycline transactivator (rtTA) gene which has been reported to be sevenfold more active and 100-fold more doxycycline sensitive than the original Tet-On system [ 8 ]. The β-globin intron ΙΙ (BGI) and poly(A) tail were incorporated into the transgene to ensure proper splicing and transcript stability, respectively. The positions of the PCR primers used for genotyping are indicated by half-arrows

Techniques Used: Expressing, Polymerase Chain Reaction

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Article Snippet: .. The full-length maa2 gene was amplified from strain H606 chromosomal DNA by PCR with primers F3 and HMPR and cloned into pGEM-T Easy as described above. .. E. coli containing recombinant plasmids with inserts in both orientations were tested for ability to express recombinant MAA2 as described for 158p10p9.

Article Title: Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics
Article Snippet: .. Amplicons of expected size were confirmed by agarose gel electrophoresis ( ) and vlhA - obg amplicon was cloned into pGEM-T Easy vector according to the manufacturer’s instructions (Promega, Alexandria, New South Wales, Australia). .. A Pst I- Sac II fragment containing vlhA - obg was excised and inserted into the compatible sites of pBluescript II KS (+) phagemid (Thermo Fisher Scientific, Scoresby, Victoria, Australia).

Agarose Gel Electrophoresis:

Article Title: Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics
Article Snippet: .. Amplicons of expected size were confirmed by agarose gel electrophoresis ( ) and vlhA - obg amplicon was cloned into pGEM-T Easy vector according to the manufacturer’s instructions (Promega, Alexandria, New South Wales, Australia). .. A Pst I- Sac II fragment containing vlhA - obg was excised and inserted into the compatible sites of pBluescript II KS (+) phagemid (Thermo Fisher Scientific, Scoresby, Victoria, Australia).

Purification:

Article Title: Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum
Article Snippet: .. Gene Cloning The PCR product was purified by PCR product purification kit (Roche) and ligated into the pGEM-T easy vector (Promega). .. The ligation reaction was transformed into DH5α (Promega) competent cells and plated on Luria-Bertani (LB) agar, containing ampicillin (50 mg/mL), 5-bromo-4 chloro-3-indolyl-β -D-galactoside (X-gal: 20 mM), and Isopropyl thio-β -D-galactoside (IPTG: 200 mg/mL).

Polymerase Chain Reaction:

Article Title: Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L)
Article Snippet: .. The amplified cDNA PCR products were cloned into pGEM-T Easy cloning vector (Promega) according to the manufacturer’s instructions. .. Freshly prepared competent cells of Escherichia coli DH5α were transformed with the recombinant plasmids.

Article Title: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection
Article Snippet: .. To further analyze CXCR4 gene disruption, the above PCR products were cloned into pGEM-T Easy vector (Promega) and sequenced. .. The indels of the CXCR4 gene were identified by comparison with the wild-type CXCR4 sequence.

Article Title: Molecular Characterization of Mycoplasma arthritidis Variable Surface Protein MAA2
Article Snippet: .. The full-length maa2 gene was amplified from strain H606 chromosomal DNA by PCR with primers F3 and HMPR and cloned into pGEM-T Easy as described above. .. E. coli containing recombinant plasmids with inserts in both orientations were tested for ability to express recombinant MAA2 as described for 158p10p9.

Article Title: Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum
Article Snippet: .. Gene Cloning The PCR product was purified by PCR product purification kit (Roche) and ligated into the pGEM-T easy vector (Promega). .. The ligation reaction was transformed into DH5α (Promega) competent cells and plated on Luria-Bertani (LB) agar, containing ampicillin (50 mg/mL), 5-bromo-4 chloro-3-indolyl-β -D-galactoside (X-gal: 20 mM), and Isopropyl thio-β -D-galactoside (IPTG: 200 mg/mL).

Plasmid Preparation:

Article Title: Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L)
Article Snippet: .. The amplified cDNA PCR products were cloned into pGEM-T Easy cloning vector (Promega) according to the manufacturer’s instructions. .. Freshly prepared competent cells of Escherichia coli DH5α were transformed with the recombinant plasmids.

Article Title: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection
Article Snippet: .. To further analyze CXCR4 gene disruption, the above PCR products were cloned into pGEM-T Easy vector (Promega) and sequenced. .. The indels of the CXCR4 gene were identified by comparison with the wild-type CXCR4 sequence.

Article Title: Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells
Article Snippet: .. M-MLV reverse transcriptase (RT), T7 RNA polymerase, GoTaq® DNA polymerase, pGEM®-T Easy vector, RNAsin® inhibitor, RNase-free DNase RQ1, and other molecular biology reagents were purchased from Promega (Madison, WI). .. RNase-free Deoxyribonuclease I (DNase I) Amplification Grade, the pcDNA3.1(+) vector, and oligonucleotides were obtained from Invitrogen (Carlsbad, CA).

Article Title: Effect of mesoporous silica under Neisseria meningitidis transformation process: environmental effects under meningococci transformation
Article Snippet: .. This fragment was cloned into the pGEM-T Easy Vector System II (Promega Corporation, Madison, WI, USA), to generate the plasmid pLAN6. .. E. coli strain Z501 was transformed with plasmid pLAN6 resulting in the plasmid pLAN7.

Article Title: Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum
Article Snippet: .. Gene Cloning The PCR product was purified by PCR product purification kit (Roche) and ligated into the pGEM-T easy vector (Promega). .. The ligation reaction was transformed into DH5α (Promega) competent cells and plated on Luria-Bertani (LB) agar, containing ampicillin (50 mg/mL), 5-bromo-4 chloro-3-indolyl-β -D-galactoside (X-gal: 20 mM), and Isopropyl thio-β -D-galactoside (IPTG: 200 mg/mL).

Article Title: Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics
Article Snippet: .. Amplicons of expected size were confirmed by agarose gel electrophoresis ( ) and vlhA - obg amplicon was cloned into pGEM-T Easy vector according to the manufacturer’s instructions (Promega, Alexandria, New South Wales, Australia). .. A Pst I- Sac II fragment containing vlhA - obg was excised and inserted into the compatible sites of pBluescript II KS (+) phagemid (Thermo Fisher Scientific, Scoresby, Victoria, Australia).

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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: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    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: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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
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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