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    Structured Review

    Thermo Fisher pcmv lacz
    ART-27 enhances steroid and thyroid receptor-dependent transcriptional activation. HeLa cells (1.2 × 10 5 cells/35-mm dish) were transiently transfected using Lipofectamine with paired receptor (0.2 μg/dish) and reporter constructs (0.1 μg/dish) for hAR + MMTV-Luc (A), GR + MMTV-Luc (B), ERα and ERβ + XETL (C), and human TRβ-1 + pGL3-DR4 (D) along with the indicated amount of ART-27 or empty expression vector to equalize the total amount of DNA per dish and 0.05 μg of <t>pCMV-LacZ</t> per dish as an internal control for transfection efficiency. Cells were treated with 100 nM R1881 (A), 100 nM dexamethasone (Dex; B), 10 nM 17β-estradiol (E2; C), 100 nM triac (T3, D), or the ethanol vehicle (white bars) for 12 h, and receptor transcriptional activation was assayed as described in MATERIALS AND METHODS, normalized to β-galactosidase activity, and expressed as relative luminescence units (RLU). The average of three independent experiments and SE is shown. (E) ART-27 does not affect transactivation by VP16. HeLa cells were transfected as above with an expression construct for GAL4-VP16 and a reporter plasmid containing five Gal4-binding sites upstream of the E1b promoter, in the absence (white bars) or presence (gray bars) of 1 μg of ART-27, and assayed for luciferase activity. (F) ART-27 failed to activate transcription when tethered to DNA. HeLa cells were transfected as above with expression constructs for LexA, LexA–ART-27, or LexA-AR 18–500 and the pΔ4X-LALO reporter plasmid and assayed for luciferase activity.
    Pcmv Lacz, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89/100 stars

    Images

    1) Product Images from "Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus"

    Article Title: Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.01-10-0513

    ART-27 enhances steroid and thyroid receptor-dependent transcriptional activation. HeLa cells (1.2 × 10 5 cells/35-mm dish) were transiently transfected using Lipofectamine with paired receptor (0.2 μg/dish) and reporter constructs (0.1 μg/dish) for hAR + MMTV-Luc (A), GR + MMTV-Luc (B), ERα and ERβ + XETL (C), and human TRβ-1 + pGL3-DR4 (D) along with the indicated amount of ART-27 or empty expression vector to equalize the total amount of DNA per dish and 0.05 μg of pCMV-LacZ per dish as an internal control for transfection efficiency. Cells were treated with 100 nM R1881 (A), 100 nM dexamethasone (Dex; B), 10 nM 17β-estradiol (E2; C), 100 nM triac (T3, D), or the ethanol vehicle (white bars) for 12 h, and receptor transcriptional activation was assayed as described in MATERIALS AND METHODS, normalized to β-galactosidase activity, and expressed as relative luminescence units (RLU). The average of three independent experiments and SE is shown. (E) ART-27 does not affect transactivation by VP16. HeLa cells were transfected as above with an expression construct for GAL4-VP16 and a reporter plasmid containing five Gal4-binding sites upstream of the E1b promoter, in the absence (white bars) or presence (gray bars) of 1 μg of ART-27, and assayed for luciferase activity. (F) ART-27 failed to activate transcription when tethered to DNA. HeLa cells were transfected as above with expression constructs for LexA, LexA–ART-27, or LexA-AR 18–500 and the pΔ4X-LALO reporter plasmid and assayed for luciferase activity.
    Figure Legend Snippet: ART-27 enhances steroid and thyroid receptor-dependent transcriptional activation. HeLa cells (1.2 × 10 5 cells/35-mm dish) were transiently transfected using Lipofectamine with paired receptor (0.2 μg/dish) and reporter constructs (0.1 μg/dish) for hAR + MMTV-Luc (A), GR + MMTV-Luc (B), ERα and ERβ + XETL (C), and human TRβ-1 + pGL3-DR4 (D) along with the indicated amount of ART-27 or empty expression vector to equalize the total amount of DNA per dish and 0.05 μg of pCMV-LacZ per dish as an internal control for transfection efficiency. Cells were treated with 100 nM R1881 (A), 100 nM dexamethasone (Dex; B), 10 nM 17β-estradiol (E2; C), 100 nM triac (T3, D), or the ethanol vehicle (white bars) for 12 h, and receptor transcriptional activation was assayed as described in MATERIALS AND METHODS, normalized to β-galactosidase activity, and expressed as relative luminescence units (RLU). The average of three independent experiments and SE is shown. (E) ART-27 does not affect transactivation by VP16. HeLa cells were transfected as above with an expression construct for GAL4-VP16 and a reporter plasmid containing five Gal4-binding sites upstream of the E1b promoter, in the absence (white bars) or presence (gray bars) of 1 μg of ART-27, and assayed for luciferase activity. (F) ART-27 failed to activate transcription when tethered to DNA. HeLa cells were transfected as above with expression constructs for LexA, LexA–ART-27, or LexA-AR 18–500 and the pΔ4X-LALO reporter plasmid and assayed for luciferase activity.

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

    2) Product Images from "The safety and longevity of DNA vaccines for fish"

    Article Title: The safety and longevity of DNA vaccines for fish

    Journal: Immunology

    doi: 10.1046/j.1365-2567.1999.00688.x

    Southern blotting. (a) 10 μg DNA from the injection site of five PCR-positive fish (fish a–e), and one control fish (CMV-0 injected muscle), were digested with Kpn I and Xba I restiction endonucleases to release a 3·2-kb lacZ fragment which was detected by a fluorescein-labelled lacZ probe in fish c, d and e. (b) Undigested DNA from differing tissues was detected by a fluorescein-labelled probe for lacZ. The injected muscle, but no other tissue, shows the three forms of plasmid DNA: linear, closed and open circles. The three samples of gut each contained a different lacZ band with a molecular weight which was distinct from that of any other sample. (c) 10 μg DNA from differing tissues was cleaved with Hin dIII and detected by the probe to the plasmid backbone. The fragment was only in the muscle and was of the same length as pCMV-lacZ.
    Figure Legend Snippet: Southern blotting. (a) 10 μg DNA from the injection site of five PCR-positive fish (fish a–e), and one control fish (CMV-0 injected muscle), were digested with Kpn I and Xba I restiction endonucleases to release a 3·2-kb lacZ fragment which was detected by a fluorescein-labelled lacZ probe in fish c, d and e. (b) Undigested DNA from differing tissues was detected by a fluorescein-labelled probe for lacZ. The injected muscle, but no other tissue, shows the three forms of plasmid DNA: linear, closed and open circles. The three samples of gut each contained a different lacZ band with a molecular weight which was distinct from that of any other sample. (c) 10 μg DNA from differing tissues was cleaved with Hin dIII and detected by the probe to the plasmid backbone. The fragment was only in the muscle and was of the same length as pCMV-lacZ.

    Techniques Used: Southern Blot, Injection, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Plasmid Preparation, Molecular Weight

    PCR analysis was used to detect the lacZ gene of pCMV-lacZ at 70 days after injection. (a) The 3·2-kb fragment was present in five of the eight fish (numbers 1, 3, 4, 5 and 7), and not in the fish injected with the empty plasmid (control fish). (b) The fragment was in the muscle but in no other tisssue from a pooled material from the three strongly positive fish.
    Figure Legend Snippet: PCR analysis was used to detect the lacZ gene of pCMV-lacZ at 70 days after injection. (a) The 3·2-kb fragment was present in five of the eight fish (numbers 1, 3, 4, 5 and 7), and not in the fish injected with the empty plasmid (control fish). (b) The fragment was in the muscle but in no other tisssue from a pooled material from the three strongly positive fish.

    Techniques Used: Polymerase Chain Reaction, Injection, Fluorescence In Situ Hybridization, Plasmid Preparation

    Counting of β-gal-positive muscle fibres at different times after intramuscular administration of pCMV-lacZ. Counts from the control fish were negative and are not shown.
    Figure Legend Snippet: Counting of β-gal-positive muscle fibres at different times after intramuscular administration of pCMV-lacZ. Counts from the control fish were negative and are not shown.

    Techniques Used: Fluorescence In Situ Hybridization

    3) Product Images from "Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells"

    Article Title: Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00469-15

    Effect of a partial double knockdown of IRSp53/Wave2 by siRNA on HIV-1 Gag localization and VLP release in T cells. (A) Jurkat T cells were transfected with the vector pGag (HIV-1 Gag expression) (a to d) or pCMV-LacZ (control) (e and f), together with a mix of siRNA (Wave2 and IRSp53) (a and f) or a mix of control siRNAs (d). The cells were fixed and stained for F-actin (phalloidin-Alexa 546) (red) and Gag (anti-MAp17-Alexa 488) (green). Merge and transmission images are presented. The cell sections show Gag and F-actin fluorescent signals at the cell periphery. (B) Distance of the maximum Gag fluorescent signal from the cell edge (determined by the actin signal) under each condition ( n = 9 to 21 cells). (C) Cell mean diameters (in micrometers) of the different cell transfection conditions, as indicated. The effect of the depletion of IRSp53 and/or Wave2 on Jurkat T-cell sizes compared to those of siRNA control-treated cells, in the presence of Gag, is shown. (D) Immunoblot analysis of pr55Gag in T-cell lysates and in VLPs. On the right are the average percentages of depletion of each protein and of inhibition of VLP release from three independent experiments. Tubulin was used as a loading control.
    Figure Legend Snippet: Effect of a partial double knockdown of IRSp53/Wave2 by siRNA on HIV-1 Gag localization and VLP release in T cells. (A) Jurkat T cells were transfected with the vector pGag (HIV-1 Gag expression) (a to d) or pCMV-LacZ (control) (e and f), together with a mix of siRNA (Wave2 and IRSp53) (a and f) or a mix of control siRNAs (d). The cells were fixed and stained for F-actin (phalloidin-Alexa 546) (red) and Gag (anti-MAp17-Alexa 488) (green). Merge and transmission images are presented. The cell sections show Gag and F-actin fluorescent signals at the cell periphery. (B) Distance of the maximum Gag fluorescent signal from the cell edge (determined by the actin signal) under each condition ( n = 9 to 21 cells). (C) Cell mean diameters (in micrometers) of the different cell transfection conditions, as indicated. The effect of the depletion of IRSp53 and/or Wave2 on Jurkat T-cell sizes compared to those of siRNA control-treated cells, in the presence of Gag, is shown. (D) Immunoblot analysis of pr55Gag in T-cell lysates and in VLPs. On the right are the average percentages of depletion of each protein and of inhibition of VLP release from three independent experiments. Tubulin was used as a loading control.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Transmission Assay, Inhibition

    Effect of siRNA-mediated depletion of Rac1 on intracellular PI(4,5)P 2 levels in Jurkat T cells. (A) Rac1-dependent cell signaling pathways having an effect on actin filament dynamics. In T cells, Rac1 GTPase plays an important role in the regulation of actin cytoskeleton rearrangement. Indeed, on one hand, in response to extracellular signals, Rac1 is activated and could stimulate actin filament turnover by the intermediate of the Pak1 (or Pak2)-LIMK-cofilin pathway. On the other hand, activated Rac1 could also stimulate actin filament branching in lamellipodia. In the latter case, IRSp53 is recruited by Rac1 and binds the proline-rich region of Wave2. As a result, Wave2 is activated and induces actin branching via Arp2/3 complex recruitment. In a third case, Rac1-GTP could also activate the phosphatidylinositol-4-phosphate 5-kinase (PIP5K), which catalyzes the synthesis of PI(4,5)P 2 at the cell plasma membrane. (B to D) Effect of siRNA-mediated depletion of Rac1 on intracellular PI(4,5)P 2 levels. (B) Cells were transfected with pCMV-LacZ, p8.2 (Gag, Gag-Pol, and viral accessory proteins), or PH-phospholipase C delta (PLCδ)-GFP, together with the siRNA control or siRNA against Rac1, as indicated. At 48 h posttransfection, cells were fixed, permeabilized, and stained with anti-MAp17 and anti-PI(4,5)P 2 antibodies. The colocalization of the GFP-tagged PH domain of phospholipase C delta and the PI(4,5)P2 antibody is indicated as a control for PI(4,5)P2-enriched membrane domains (white arrows). (C) The ratio of the PI(4,5)P 2 signal intensity to the cell area was measured by image analysis (ImageJ) ( n = 20 cells). (D) Immunoblot analysis of Rac1 GTPase and HIV-1 Gag in cell lysates and/or in virus particles. Tubulin was used as a loading control.
    Figure Legend Snippet: Effect of siRNA-mediated depletion of Rac1 on intracellular PI(4,5)P 2 levels in Jurkat T cells. (A) Rac1-dependent cell signaling pathways having an effect on actin filament dynamics. In T cells, Rac1 GTPase plays an important role in the regulation of actin cytoskeleton rearrangement. Indeed, on one hand, in response to extracellular signals, Rac1 is activated and could stimulate actin filament turnover by the intermediate of the Pak1 (or Pak2)-LIMK-cofilin pathway. On the other hand, activated Rac1 could also stimulate actin filament branching in lamellipodia. In the latter case, IRSp53 is recruited by Rac1 and binds the proline-rich region of Wave2. As a result, Wave2 is activated and induces actin branching via Arp2/3 complex recruitment. In a third case, Rac1-GTP could also activate the phosphatidylinositol-4-phosphate 5-kinase (PIP5K), which catalyzes the synthesis of PI(4,5)P 2 at the cell plasma membrane. (B to D) Effect of siRNA-mediated depletion of Rac1 on intracellular PI(4,5)P 2 levels. (B) Cells were transfected with pCMV-LacZ, p8.2 (Gag, Gag-Pol, and viral accessory proteins), or PH-phospholipase C delta (PLCδ)-GFP, together with the siRNA control or siRNA against Rac1, as indicated. At 48 h posttransfection, cells were fixed, permeabilized, and stained with anti-MAp17 and anti-PI(4,5)P 2 antibodies. The colocalization of the GFP-tagged PH domain of phospholipase C delta and the PI(4,5)P2 antibody is indicated as a control for PI(4,5)P2-enriched membrane domains (white arrows). (C) The ratio of the PI(4,5)P 2 signal intensity to the cell area was measured by image analysis (ImageJ) ( n = 20 cells). (D) Immunoblot analysis of Rac1 GTPase and HIV-1 Gag in cell lysates and/or in virus particles. Tubulin was used as a loading control.

    Techniques Used: Transfection, Staining

    4) Product Images from "Brain-Derived Neurotrophic Factor Signaling Rewrites the Glucocorticoid Transcriptome via Glucocorticoid Receptor Phosphorylation"

    Article Title: Brain-Derived Neurotrophic Factor Signaling Rewrites the Glucocorticoid Transcriptome via Glucocorticoid Receptor Phosphorylation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00150-13

    BDNF signaling enhances GR transcriptional activation. (A) HEK-293 cells ectopically expressing TrkB (293TrkB) were transiently transfected with pCMV-rat GR, the TAT3-Luc reporter construct, and pCMV-lacZ as an internal control. Twenty-four hours after
    Figure Legend Snippet: BDNF signaling enhances GR transcriptional activation. (A) HEK-293 cells ectopically expressing TrkB (293TrkB) were transiently transfected with pCMV-rat GR, the TAT3-Luc reporter construct, and pCMV-lacZ as an internal control. Twenty-four hours after

    Techniques Used: Activation Assay, Expressing, Transfection, Construct

    5) Product Images from "Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c"

    Article Title: Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c

    Journal: Molecular and Cellular Biology

    doi:

    Deletion analysis of hILP. (A) Schematic diagram of hILP showing deletion mutants. BIR domains (solid boxes), the amphipathic region (open box), and the ring finger (hatched box) are shown. WT, wild type. (B) MCF7F cells were transfected with pCMV-lacZ and either pEBB, pEBB-hILP, or pEBB deletion mutants of hILP. After 24 h, medium was removed and replaced with either fresh medium alone or fresh medium plus rhTNF-α. Cells were incubated for 12 h, at which time cells were fixed, stained for β-galactosidase expression, and scored for apoptotic morphology. The expression of FLAG-tagged deletion constructs was confirmed by immunoblotting with anti-FLAG bioM5 (Kodak) (data not shown). Results are expressed as percent viable cells (number of flat blue cells/number of flat and round blue cells × 100). The data represent the means ± standard deviations ( n = 3).
    Figure Legend Snippet: Deletion analysis of hILP. (A) Schematic diagram of hILP showing deletion mutants. BIR domains (solid boxes), the amphipathic region (open box), and the ring finger (hatched box) are shown. WT, wild type. (B) MCF7F cells were transfected with pCMV-lacZ and either pEBB, pEBB-hILP, or pEBB deletion mutants of hILP. After 24 h, medium was removed and replaced with either fresh medium alone or fresh medium plus rhTNF-α. Cells were incubated for 12 h, at which time cells were fixed, stained for β-galactosidase expression, and scored for apoptotic morphology. The expression of FLAG-tagged deletion constructs was confirmed by immunoblotting with anti-FLAG bioM5 (Kodak) (data not shown). Results are expressed as percent viable cells (number of flat blue cells/number of flat and round blue cells × 100). The data represent the means ± standard deviations ( n = 3).

    Techniques Used: Transfection, Incubation, Staining, Expressing, Construct

    hILP blocks cell death induced by a variety of stimuli. (A) MCF7F cells were transfected with pEBB ( ), pEBB-hILP (▪), pCI (□), or pCI-Bcl-x L (▨). Twenty-four hours after transfection, cells were treated with fresh medium or medium supplemented with either anti-Fas antibody and cycloheximide, rhTNF-α, or cisplatin or were exposed to UV. After 12 h, cells were fixed, stained for β-galactosidase expression, and scored for apoptotic morphology. (B) MCF7F cells were transfected with pCMV-lacZ together with pcDNA3, pcDNA3-caspase 2, or pcDNA3-caspase 8 with or without cotransfection of pEBB-hILP expression vector. At 24 h after transfection, cells were fixed, stained for β-galactosidase expression, and scored for apoptotic morphology. Results are expressed as percent viable cells (number of flat blue cells/number of flat and round blue cells × 100). The data represent means ± standard deviations ( n = 3).
    Figure Legend Snippet: hILP blocks cell death induced by a variety of stimuli. (A) MCF7F cells were transfected with pEBB ( ), pEBB-hILP (▪), pCI (□), or pCI-Bcl-x L (▨). Twenty-four hours after transfection, cells were treated with fresh medium or medium supplemented with either anti-Fas antibody and cycloheximide, rhTNF-α, or cisplatin or were exposed to UV. After 12 h, cells were fixed, stained for β-galactosidase expression, and scored for apoptotic morphology. (B) MCF7F cells were transfected with pCMV-lacZ together with pcDNA3, pcDNA3-caspase 2, or pcDNA3-caspase 8 with or without cotransfection of pEBB-hILP expression vector. At 24 h after transfection, cells were fixed, stained for β-galactosidase expression, and scored for apoptotic morphology. Results are expressed as percent viable cells (number of flat blue cells/number of flat and round blue cells × 100). The data represent means ± standard deviations ( n = 3).

    Techniques Used: Transfection, Staining, Expressing, Cotransfection, Plasmid Preparation

    Related Articles

    Transfection:

    Article Title: Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus
    Article Snippet: .. For transfections, HeLa cells were seeded in 35-mm dishes at a density of 1.3 × 105 , washed once with serum-free medium, and transfected with 0.2 μg of pcDNA3:human AR, 0.1 μg of pMMTV-Luc, 0.05 μg of pCMV-LacZ, and the indicated amounts of pcDNA3:HA–ART-27 using 5 μl of Lipofectamine reagent (Invitrogen) in a total volume of 1 ml of serum-free, phenol red-free DMEM per 35-mm dish according to the manufacturer's instructions. .. Approximately 4 h posttransfection, the transfection mixture was removed, and the cells were refed with 2 ml of DMEM-10% FBS, allowed to recover for 3–5 h, and fed again with fresh DMEM-10% FBS supplemented with 100 nM R1881, dexamethasone, 17β-estradiol, triac, or an identical volume of 100% ethanol and incubated for 12 h. Transfected cells were washed once in phosphate-buffered saline (PBS) and harvested in 1X reporter lysis buffer (Promega) according to the manufacturer's instructions.

    Article Title: Brain-Derived Neurotrophic Factor Signaling Rewrites the Glucocorticoid Transcriptome via Glucocorticoid Receptor Phosphorylation
    Article Snippet: .. Transfections of HEK-293 and 293TrkB cells with TAT3-LUC reporter plasmid, pCMV-LacZ, pCMV-rat GR, or FCIV1-rat GR were performed using Lipofectamine 2000 (Invitrogen). .. Electroporations of neurons were performed using FCIV1-rat GR and GR S155A/S287A with an Amaxa rat neuron Nucleofector kit (Lonza) according to the manufacturer's instructions.

    Article Title: Role of Promyelocytic Leukemia Zinc Finger (PLZF) in Cell Proliferation and Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) Gene Repression *
    Article Snippet: .. The reporter fusion plasmids pGL2-ARF-Luc, pGL2-MDM2-Luc, pGL2-TP53-Luc, pG13-Luc, pG5–5x(GC-box)-Luc, and pGL2-CDKN1A-Luc, as well as pcDNA3.1-PLZF, pcDNA3-p53, and pCMV-LacZ in various combinations were transiently transfected into cell lines using Lipofectamine Plus reagent (Invitrogen) and analyzed as described elsewhere ( , ) .. The following two siRNA against PLZF mRNA were designed and purchased from Bioneer (Taejeon, Korea): PLZF siRNA-1, sense, 5′-GGC CAACCAGAUGCGGCUGUU-3′, and antisense, 5′-UUCCGGUUGGUCUACGCCGAC-3′; and PLZF siRNA-2, sense, 5′-GAUGUUUGAGAUCCUCUUCUU-3′, and antisense, 5′-UUCUACAAACUCUAGGAGAAG-3′.

    Article Title: Hypoxic metabolism in human hematopoietic stem cells
    Article Snippet: .. 0.8 μg of Hif-1α-pGL2 or PbxMut-Hif-1α-pGL2 or HoxMut-Hif-1α-pGL2 reporter construct were co-transfectedwith different doses of the Meis1, Meis1∆PIM, HoxA9, Hoxa∆MID and Pbx1 expression vectors and 0.2 μg of pCMV-LacZ (internal control) into COS cells using lipofectamine transfection reagent (Invitrogen). .. At 48 h after transfection, cell lysate were prepared and quantified for firefly luciferase activity using a luciferase reporter system (Promega).

    Article Title: GAS41 interacts with transcription factor AP-2? and stimulates AP-2?-mediated transactivation
    Article Snippet: .. Luciferase assays Transient transfections of cells with A2-Luc, pCMV-LacZ and the indicated expression vectors were carried out with Lipofectamine 2000 (Invitrogen). .. Twenty four hours after transfection, the cells were lysed and luciferase assay was performed using the luciferase assay system (Promega). pCMV-LacZ was cotransfected in all experiments, and β-galactosidase activity was used to normalize for different transfection efficiencies.

    Luciferase:

    Article Title: GAS41 interacts with transcription factor AP-2? and stimulates AP-2?-mediated transactivation
    Article Snippet: .. Luciferase assays Transient transfections of cells with A2-Luc, pCMV-LacZ and the indicated expression vectors were carried out with Lipofectamine 2000 (Invitrogen). .. Twenty four hours after transfection, the cells were lysed and luciferase assay was performed using the luciferase assay system (Promega). pCMV-LacZ was cotransfected in all experiments, and β-galactosidase activity was used to normalize for different transfection efficiencies.

    Plasmid Preparation:

    Article Title: Brain-Derived Neurotrophic Factor Signaling Rewrites the Glucocorticoid Transcriptome via Glucocorticoid Receptor Phosphorylation
    Article Snippet: .. Transfections of HEK-293 and 293TrkB cells with TAT3-LUC reporter plasmid, pCMV-LacZ, pCMV-rat GR, or FCIV1-rat GR were performed using Lipofectamine 2000 (Invitrogen). .. Electroporations of neurons were performed using FCIV1-rat GR and GR S155A/S287A with an Amaxa rat neuron Nucleofector kit (Lonza) according to the manufacturer's instructions.

    Article Title: The safety and longevity of DNA vaccines for fish
    Article Snippet: .. The pCMV-lacZ was pcDNA3.1/His/LacZ plasmid (Invitrogen, Leek, The Netherlands). .. The control plasmid (pCMV-0) was pcDNA3 (Invitrogen).

    Article Title: Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c
    Article Snippet: .. A 1:5 ratio of pCMV-lacZ or pGreenLantern (pCMV-GLP; Life Technologies) to test expression vector was used to ensure that every β-galactosidase- or GreenLantern protein (GLP)-positive cell had also taken up the test expression vector. .. For apoptosis assays, MCF7 cells were treated with either anti-Fas (150 ng/ml) plus cycloheximide (1 μg/ml), recombinant human TNF-α (rhTNF-α) (40 ng/ml), cisplatin (20 μg/ml), or UV light (5 min on a 312-nm transilluminator [Fisherbiotech]).

    Construct:

    Article Title: Hypoxic metabolism in human hematopoietic stem cells
    Article Snippet: .. 0.8 μg of Hif-1α-pGL2 or PbxMut-Hif-1α-pGL2 or HoxMut-Hif-1α-pGL2 reporter construct were co-transfectedwith different doses of the Meis1, Meis1∆PIM, HoxA9, Hoxa∆MID and Pbx1 expression vectors and 0.2 μg of pCMV-LacZ (internal control) into COS cells using lipofectamine transfection reagent (Invitrogen). .. At 48 h after transfection, cell lysate were prepared and quantified for firefly luciferase activity using a luciferase reporter system (Promega).

    Expressing:

    Article Title: Hypoxic metabolism in human hematopoietic stem cells
    Article Snippet: .. 0.8 μg of Hif-1α-pGL2 or PbxMut-Hif-1α-pGL2 or HoxMut-Hif-1α-pGL2 reporter construct were co-transfectedwith different doses of the Meis1, Meis1∆PIM, HoxA9, Hoxa∆MID and Pbx1 expression vectors and 0.2 μg of pCMV-LacZ (internal control) into COS cells using lipofectamine transfection reagent (Invitrogen). .. At 48 h after transfection, cell lysate were prepared and quantified for firefly luciferase activity using a luciferase reporter system (Promega).

    Article Title: Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c
    Article Snippet: .. A 1:5 ratio of pCMV-lacZ or pGreenLantern (pCMV-GLP; Life Technologies) to test expression vector was used to ensure that every β-galactosidase- or GreenLantern protein (GLP)-positive cell had also taken up the test expression vector. .. For apoptosis assays, MCF7 cells were treated with either anti-Fas (150 ng/ml) plus cycloheximide (1 μg/ml), recombinant human TNF-α (rhTNF-α) (40 ng/ml), cisplatin (20 μg/ml), or UV light (5 min on a 312-nm transilluminator [Fisherbiotech]).

    Article Title: GAS41 interacts with transcription factor AP-2? and stimulates AP-2?-mediated transactivation
    Article Snippet: .. Luciferase assays Transient transfections of cells with A2-Luc, pCMV-LacZ and the indicated expression vectors were carried out with Lipofectamine 2000 (Invitrogen). .. Twenty four hours after transfection, the cells were lysed and luciferase assay was performed using the luciferase assay system (Promega). pCMV-LacZ was cotransfected in all experiments, and β-galactosidase activity was used to normalize for different transfection efficiencies.

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    Thermo Fisher pcmv lacz vector
    CDK9–cyclin T1 complex regulates the recruitment of ATF4 to the CHOP promoter under ER stress. A, HEK293T cells were co-transfected with firefly luciferase reporter plasmid containing three repeats of the C/EBP–ATF composite (−303/−292) of the mouse CHOP gene, <t>pCMV-LacZ,</t> and control or human ATF4 expression plasmid. 4 h after transfection; cells were incubated in DMEM containing 10% fetal bovine serum with DMSO ( Veh ) or CDK9 inhibitors ( CDK9i ) such as 300 n m flavopiridol or 30 μ m CAY10574 for 24 h. The results are expressed as luciferase ( Luc )/β-gal units of induction ( n -fold) over the control value for each construct. One-way ANOVA with a Student-Newman post hoc test was used for comparison between vehicle, flavopiridol, and CAY10574-treated cells ( n = 6). B, ChIP analysis of SFA-induced ATF4 recruitment to the C/EBP-ATF composite of the CHOP promoter. VSMCs were pretreated with 10 μ m flavopiridol (CDK9i) for 2 h, and then treated with BSA ( Veh ) or 250 μ m C18:0 with or without 10 μ m CDK9i. After 6 h, chromatin fractions of VSMCs were prepared. ChIP was performed with rabbit control IgG or ATF4 antibodies. Purified immunoprecipitated DNA was analyzed by qRT-PCR with primers for the C/EBP–ATF composite of the CHOP promoter. The results are expressed as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin (% input) ( n = 3). One-way ANOVA with a Student-Newman post hoc test was used for statistical analysis. *, p
    Pcmv Lacz Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv lacz vector/product/Thermo Fisher
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    CDK9–cyclin T1 complex regulates the recruitment of ATF4 to the CHOP promoter under ER stress. A, HEK293T cells were co-transfected with firefly luciferase reporter plasmid containing three repeats of the C/EBP–ATF composite (−303/−292) of the mouse CHOP gene, pCMV-LacZ, and control or human ATF4 expression plasmid. 4 h after transfection; cells were incubated in DMEM containing 10% fetal bovine serum with DMSO ( Veh ) or CDK9 inhibitors ( CDK9i ) such as 300 n m flavopiridol or 30 μ m CAY10574 for 24 h. The results are expressed as luciferase ( Luc )/β-gal units of induction ( n -fold) over the control value for each construct. One-way ANOVA with a Student-Newman post hoc test was used for comparison between vehicle, flavopiridol, and CAY10574-treated cells ( n = 6). B, ChIP analysis of SFA-induced ATF4 recruitment to the C/EBP-ATF composite of the CHOP promoter. VSMCs were pretreated with 10 μ m flavopiridol (CDK9i) for 2 h, and then treated with BSA ( Veh ) or 250 μ m C18:0 with or without 10 μ m CDK9i. After 6 h, chromatin fractions of VSMCs were prepared. ChIP was performed with rabbit control IgG or ATF4 antibodies. Purified immunoprecipitated DNA was analyzed by qRT-PCR with primers for the C/EBP–ATF composite of the CHOP promoter. The results are expressed as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin (% input) ( n = 3). One-way ANOVA with a Student-Newman post hoc test was used for statistical analysis. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The CDK9–cyclin T1 complex mediates saturated fatty acid–induced vascular calcification by inducing expression of the transcription factor CHOP

    doi: 10.1074/jbc.RA118.004706

    Figure Lengend Snippet: CDK9–cyclin T1 complex regulates the recruitment of ATF4 to the CHOP promoter under ER stress. A, HEK293T cells were co-transfected with firefly luciferase reporter plasmid containing three repeats of the C/EBP–ATF composite (−303/−292) of the mouse CHOP gene, pCMV-LacZ, and control or human ATF4 expression plasmid. 4 h after transfection; cells were incubated in DMEM containing 10% fetal bovine serum with DMSO ( Veh ) or CDK9 inhibitors ( CDK9i ) such as 300 n m flavopiridol or 30 μ m CAY10574 for 24 h. The results are expressed as luciferase ( Luc )/β-gal units of induction ( n -fold) over the control value for each construct. One-way ANOVA with a Student-Newman post hoc test was used for comparison between vehicle, flavopiridol, and CAY10574-treated cells ( n = 6). B, ChIP analysis of SFA-induced ATF4 recruitment to the C/EBP-ATF composite of the CHOP promoter. VSMCs were pretreated with 10 μ m flavopiridol (CDK9i) for 2 h, and then treated with BSA ( Veh ) or 250 μ m C18:0 with or without 10 μ m CDK9i. After 6 h, chromatin fractions of VSMCs were prepared. ChIP was performed with rabbit control IgG or ATF4 antibodies. Purified immunoprecipitated DNA was analyzed by qRT-PCR with primers for the C/EBP–ATF composite of the CHOP promoter. The results are expressed as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin (% input) ( n = 3). One-way ANOVA with a Student-Newman post hoc test was used for statistical analysis. *, p

    Article Snippet: Cells were transfected with 200 ng of firefly luciferase reporter plasmids (pGL3, Promega), 50 ng of expression plasmid (FLAG-Atf4-pcDNA3.1), and 100 ng of pCMV-LacZ vector using Turbofect (ThermoFisher Scientific).

    Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Incubation, Construct, Chromatin Immunoprecipitation, Purification, Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Polymerase Chain Reaction