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  • 98
    Thermo Fisher pcdna5 flag
    Efficiency of stable cell line generation. (A) Influence of plasmid quantity and selection stringency on the number of colonies obtained following stable transfection of 293 Flp-In T-REx cells. 1.0 μg of pOG44 was mixed with the indicated amounts of <t>pcDNA5/FRT/TO</t> and used for transfection. Cells were selected by treatment with the indicated concentration of hygromycin B and constant concentration of blasticidin S (10 μg/ml). Colonies were stained with crystal violet. (B) Comparison of stable transfection efficiency with pcDNA5/FRT/TO or its pKK derivatives. Cells were transfected with 300 ng of indicated plasmids and 1.0 μg of pOG44 and subjected to selection with hygromycin B (50 μg/ml) and blasticidin S (10 μg/ml).
    Pcdna5 Flag, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher pcdna5 frt
    Western blot analysis of antibody expression in single cell clones. The selected single stable-cell clones were expanded in selection medium which was replaced with serum-free medium. Day 4 serum-free conditioned medium (CM) from each clone was collected and analyzed by SDS-PAGE. After electrophoretic separation, the proteins were transferred onto PVDF membrane, stained by anti human-Fc monoclonal antibody and visualized by SEC technology (Amersham). (A) FCHO cell clones stably transfected by a mixture of both vectors FV and <t>pcDNA5/FRT-Fc-Fusion.</t> (B) Regular CHO cell clones stably transfected by vector pcDNA5/FRT-FC-Fusion. (C) FCHO cell clones stably transfected by vector pcDNA5/FRT-FC-Fusion.
    Pcdna5 Frt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher pcdna5 to plasmid
    CUL5-NEDD8 conjugation is required for 17-AAG–induced client degradation but not for loss of client activity. HT29 human colon cancer cells were transfected with <t>pcDNA5/FRT/TO</t> plasmids containing HA-tagged CUL5 (CUL5-WT), an HA-tagged CUL5 triple
    Pcdna5 To Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher pcdna5 to expression vector
    Depletion of Wdr82 decreases Setd1A occupancy and histone H3-Lys4 trimethylation near the transcription start site. A. Diagram of the inducible Wdr82 miRNA construct. Two 118-nt modified miR-30 hairpins for Wdr82 were tandemly subcloned into the <t>pcDNA5/TO</t> vector, which contains a C-terminal green fluorescent protein cDNA and is expressed under control of a tetracycline operator. Restriction enzyme sites used for subcloning are underlined. B. Inducible T-REx HEK293 cell lines that express tandem miRNA of human Wdr82, tandem scramble miRNA, or vector only were induced for 5 days with 1 μg/ml doxycycline. Nuclear extracts were prepared and analyzed by Western blotting using the indicated antisera. The results are shown for two independent clones that express the tandem miRNA of human Wdr82. C. Cell lines that express the tandem miRNA of Wdr82 were induced for 5 days with 1 μg/ml doxycycline. Histone H3-Lys4 methylation near the transcription start site of the PPIA , PABPC1 , and GAPDH genes and the promoter region of the HOXC8 gene were analyzed by ChIP and real-time PCR. The level of histone H3-Lys4 methylation in uninduced cells was set at 100%. The data represent a summary of three independent experiments. Error bars indicate the mean standard deviations, and P values were determined by a standard t test. Asterisks indicate P values of
    Pcdna5 To Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Efficiency of stable cell line generation. (A) Influence of plasmid quantity and selection stringency on the number of colonies obtained following stable transfection of 293 Flp-In T-REx cells. 1.0 μg of pOG44 was mixed with the indicated amounts of pcDNA5/FRT/TO and used for transfection. Cells were selected by treatment with the indicated concentration of hygromycin B and constant concentration of blasticidin S (10 μg/ml). Colonies were stained with crystal violet. (B) Comparison of stable transfection efficiency with pcDNA5/FRT/TO or its pKK derivatives. Cells were transfected with 300 ng of indicated plasmids and 1.0 μg of pOG44 and subjected to selection with hygromycin B (50 μg/ml) and blasticidin S (10 μg/ml).

    Journal: PLoS ONE

    Article Title: Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

    doi: 10.1371/journal.pone.0194887

    Figure Lengend Snippet: Efficiency of stable cell line generation. (A) Influence of plasmid quantity and selection stringency on the number of colonies obtained following stable transfection of 293 Flp-In T-REx cells. 1.0 μg of pOG44 was mixed with the indicated amounts of pcDNA5/FRT/TO and used for transfection. Cells were selected by treatment with the indicated concentration of hygromycin B and constant concentration of blasticidin S (10 μg/ml). Colonies were stained with crystal violet. (B) Comparison of stable transfection efficiency with pcDNA5/FRT/TO or its pKK derivatives. Cells were transfected with 300 ng of indicated plasmids and 1.0 μg of pOG44 and subjected to selection with hygromycin B (50 μg/ml) and blasticidin S (10 μg/ml).

    Article Snippet: Vector construction pKK and pKK-RNAtag vectors were constructed by modifying pcDNA5/FRT/TO (Thermo Fisher Scientific); pKK-BI16, pKK-RNAi, pKK-BiFC and pKK-FRET vectors were constructed by modifying BI16 (a kind gift from Ed Grabczyk), which is in turn derived from pcDNA5/FRT/TO [ ].

    Techniques: Stable Transfection, Plasmid Preparation, Selection, Transfection, Concentration Assay, Staining

    Expression of APP in wild-type or mutant APP -transfected cells. (A) The distribution of APP was similar in APP -transfected cells. The scale bar represents 50 μm. (B) The expression of total APP and β-actin in cell lysates (5 μg protein/lane) was detected by immunoblotting. Full-sized images are available in Supplementary Material. The summed density of the two bands (arrows) was quantified as the amount of total APP and demonstrated in (C). Nc: negative control, Vec: pcDNA5/FRT/TO vector-transfected cells, Wt: wild-type, Sw: Swedish, Du: Dutch, Lo: London-type mutant-transfected cells, respectively, a.u.: arbitrary unit.

    Journal: Heliyon

    Article Title: Mutations in the β-amyloid precursor protein in familial Alzheimer’s disease increase Aβ oligomer production in cellular models

    doi: 10.1016/j.heliyon.2018.e00511

    Figure Lengend Snippet: Expression of APP in wild-type or mutant APP -transfected cells. (A) The distribution of APP was similar in APP -transfected cells. The scale bar represents 50 μm. (B) The expression of total APP and β-actin in cell lysates (5 μg protein/lane) was detected by immunoblotting. Full-sized images are available in Supplementary Material. The summed density of the two bands (arrows) was quantified as the amount of total APP and demonstrated in (C). Nc: negative control, Vec: pcDNA5/FRT/TO vector-transfected cells, Wt: wild-type, Sw: Swedish, Du: Dutch, Lo: London-type mutant-transfected cells, respectively, a.u.: arbitrary unit.

    Article Snippet: In order to introduce human wild-type APP 695 cDNA and Swedish-mutant APP 695 cDNA (K670NM671L) into a pcDNA5/FRT/TO vector (Invitrogen), pEF-BOS vectors harboring wild- or Swedish-type APP (gifted from Dementia and Higher Brain Function Research, Tokyo Metropolitan Institute of Medical Science) were used as templates to amplify APP cDNA with or without mutations by PCR.

    Techniques: Expressing, Mutagenesis, Transfection, Negative Control, Plasmid Preparation

    Generating stable, inducible INS-1 cell lines by Flp recombinase-mediated integration. The generation of the Flp-In T-REx cells involves three integration steps that are schematically illustrated. In the first step, the Flp recognition target (FRT) is integrated by transfection of the FRT/lacZeo2 plasmid. The use of the lacZ–Zeocin fusion gene allows selection for Zeocin resistant cell clones and the lacZ portion is used to characterize the integration site by scoring the β-galactosidase activity. In the second step, an expression vector (pcDNA6/TR) encoding the tetracycline repressor (TetR) is introduced using blasticidin resistance. In the third step, the GOI (gene-of-interest) is introduced by Flp recombinase activity from the pCSFLPe plasmid using the pcDNA5/FRT/TO vector containing the GOI. This construct contains an FRT site in front of the hygromycin ORF that will generate hygromycin resistance only, if a site-directed integration occurs at the FRT site downstream of the ATG of the lacZ–Zeocin cassette. By this recombination, the GOI present in the pcDNA5/FRT/TO vector is integrated downstream of the FRT integration site. As the GOI is driven by the tetracycline operator controlled CMV promoter (P CMV/2TetO2 ), tetracycline regulation can be obtained. P SV40 (SV40 promoter), Amp (ampiciline resistance), pUCori (bacterial origin of replication), P CMV (CMV promoter), SV40pA (SV40 polyadenylation), BGHpA (bovine growth hormone polyadenylation site). For further details see Flp-In™ system manual (Invitrogen).

    Journal: Nucleic Acids Research

    Article Title: Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4? and HNF1? in a pancreatic ?-cell line

    doi: 10.1093/nar/gnh144

    Figure Lengend Snippet: Generating stable, inducible INS-1 cell lines by Flp recombinase-mediated integration. The generation of the Flp-In T-REx cells involves three integration steps that are schematically illustrated. In the first step, the Flp recognition target (FRT) is integrated by transfection of the FRT/lacZeo2 plasmid. The use of the lacZ–Zeocin fusion gene allows selection for Zeocin resistant cell clones and the lacZ portion is used to characterize the integration site by scoring the β-galactosidase activity. In the second step, an expression vector (pcDNA6/TR) encoding the tetracycline repressor (TetR) is introduced using blasticidin resistance. In the third step, the GOI (gene-of-interest) is introduced by Flp recombinase activity from the pCSFLPe plasmid using the pcDNA5/FRT/TO vector containing the GOI. This construct contains an FRT site in front of the hygromycin ORF that will generate hygromycin resistance only, if a site-directed integration occurs at the FRT site downstream of the ATG of the lacZ–Zeocin cassette. By this recombination, the GOI present in the pcDNA5/FRT/TO vector is integrated downstream of the FRT integration site. As the GOI is driven by the tetracycline operator controlled CMV promoter (P CMV/2TetO2 ), tetracycline regulation can be obtained. P SV40 (SV40 promoter), Amp (ampiciline resistance), pUCori (bacterial origin of replication), P CMV (CMV promoter), SV40pA (SV40 polyadenylation), BGHpA (bovine growth hormone polyadenylation site). For further details see Flp-In™ system manual (Invitrogen).

    Article Snippet: The Flp expression vector used (pCSFLPe) has been described previously , whereas the pFRT/l ac Zeo2, pcDNA6/TR and pcDNA5/FRT/TO vectors were obtained from Invitrogen.

    Techniques: Transfection, Plasmid Preparation, Selection, Clone Assay, Activity Assay, Expressing, Construct

    Western blot analysis of antibody expression in single cell clones. The selected single stable-cell clones were expanded in selection medium which was replaced with serum-free medium. Day 4 serum-free conditioned medium (CM) from each clone was collected and analyzed by SDS-PAGE. After electrophoretic separation, the proteins were transferred onto PVDF membrane, stained by anti human-Fc monoclonal antibody and visualized by SEC technology (Amersham). (A) FCHO cell clones stably transfected by a mixture of both vectors FV and pcDNA5/FRT-Fc-Fusion. (B) Regular CHO cell clones stably transfected by vector pcDNA5/FRT-FC-Fusion. (C) FCHO cell clones stably transfected by vector pcDNA5/FRT-FC-Fusion.

    Journal: mAbs

    Article Title: Development of a novel mammalian cell surface antibody display platform

    doi: 10.4161/mabs.2.5.12970

    Figure Lengend Snippet: Western blot analysis of antibody expression in single cell clones. The selected single stable-cell clones were expanded in selection medium which was replaced with serum-free medium. Day 4 serum-free conditioned medium (CM) from each clone was collected and analyzed by SDS-PAGE. After electrophoretic separation, the proteins were transferred onto PVDF membrane, stained by anti human-Fc monoclonal antibody and visualized by SEC technology (Amersham). (A) FCHO cell clones stably transfected by a mixture of both vectors FV and pcDNA5/FRT-Fc-Fusion. (B) Regular CHO cell clones stably transfected by vector pcDNA5/FRT-FC-Fusion. (C) FCHO cell clones stably transfected by vector pcDNA5/FRT-FC-Fusion.

    Article Snippet: Flp-vector (FV, ) was constructed by inserting two expression cassettes into pcDNA5-FRT (Invitrogen) for the expression of the secreted version of full-length antibody.

    Techniques: Western Blot, Expressing, Clone Assay, Stable Transfection, Selection, SDS Page, Staining, Size-exclusion Chromatography, Transfection, Plasmid Preparation

    Schematic illustration of Homologous Recombination by the Flp-In ™ System. Two expression cassettes for heavy and light chains were inserted into pcDNA5/FRT to form vector FV. Vector pOG44 contained the Flp recombinase gene. After co-transfection of both vectors FV and pOG44, the Flp recombinase expressed from pOG44 catalyzes a homologous recombination event between the FRT sites of the Flp-In host cell and the vector FV. Then the whole vector is inserted by homologous integration into the genome of the host cell only at the location of FRT.

    Journal: mAbs

    Article Title: Development of a novel mammalian cell surface antibody display platform

    doi: 10.4161/mabs.2.5.12970

    Figure Lengend Snippet: Schematic illustration of Homologous Recombination by the Flp-In ™ System. Two expression cassettes for heavy and light chains were inserted into pcDNA5/FRT to form vector FV. Vector pOG44 contained the Flp recombinase gene. After co-transfection of both vectors FV and pOG44, the Flp recombinase expressed from pOG44 catalyzes a homologous recombination event between the FRT sites of the Flp-In host cell and the vector FV. Then the whole vector is inserted by homologous integration into the genome of the host cell only at the location of FRT.

    Article Snippet: Flp-vector (FV, ) was constructed by inserting two expression cassettes into pcDNA5-FRT (Invitrogen) for the expression of the secreted version of full-length antibody.

    Techniques: Homologous Recombination, Expressing, Plasmid Preparation, Cotransfection

    TRAIL expression of cell line JB6-pcDNA5/FRT TRAIL . The lacZeo expression cassette was exchanged for the full-length cDNA of TRAIL by FRT recombination. The resulting cell clone was examined for TRAIL expression. a Western Blot of the cellular lysates of JB6pcDNA5/FRT TRAIL and JB6pcDNA5/FRT CAT. Immunostaining was performed by treatment with anti-TRAIL followed by incubation with alkaline phosphatase conjugated secondary antibody and colour development of NBT/BCIP. b Indirect immunofluorescence of JB6pcDNA5/FRT TRAIL. Cells were fixated and incubated with anti-TRAIL either directed against the C terminus or directed against the N terminus. The intracellular domain is detected only if the cells are permeable.

    Journal: Annals of Surgical Innovation and Research

    Article Title: Expression of TNF-related apoptosis-inducing ligand (TRAIL) in keratinocytes mediates apoptotic cell death in allogenic T cells

    doi: 10.1186/1750-1164-3-13

    Figure Lengend Snippet: TRAIL expression of cell line JB6-pcDNA5/FRT TRAIL . The lacZeo expression cassette was exchanged for the full-length cDNA of TRAIL by FRT recombination. The resulting cell clone was examined for TRAIL expression. a Western Blot of the cellular lysates of JB6pcDNA5/FRT TRAIL and JB6pcDNA5/FRT CAT. Immunostaining was performed by treatment with anti-TRAIL followed by incubation with alkaline phosphatase conjugated secondary antibody and colour development of NBT/BCIP. b Indirect immunofluorescence of JB6pcDNA5/FRT TRAIL. Cells were fixated and incubated with anti-TRAIL either directed against the C terminus or directed against the N terminus. The intracellular domain is detected only if the cells are permeable.

    Article Snippet: Generation of TRAIL producing cell clones Full length TRAIL cDNA was cloned from a human lymphoma cDNA library (Lym12, Invitrogen, CA) using -ATG GCT ATG ATG GAG GTC- as 5' and -TTA GCC AAC TAA AAA GGC- as 3' primer, according to the published sequence (GeneBank accession number: U37518 ), and cloned into the expression vector pcDNA5/FRT (Invitrogen).

    Techniques: Expressing, Western Blot, Immunostaining, Incubation, Immunofluorescence

    The effect of TRAIL expression in JB6-pcDNA5/FRT TRAIL cells on co-cultured Jurkat cells . A Phase-contrast-imaging of apoptotic Jurkat cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. b Annexin V/Propidiumiodide dyed Jurkat cells after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. Membrane fluorescence is characteristic for apoptosis. c Phase-contrast-imaging of a Jurkat cell after 48 hours of co-culture with JB6-pcDNA5/FRT CAT. d Annexin V/Propidiumiodide dyed Jurkat cell after 48 hours of co-culture with JB6-pcDNA5/FRT CAT. No membrane fluorescence could be detected. e Western blot stained with anti-Caspase 8 followed by alkaline phosphatase-conjugated secondary antibody and NBT/BCIP colour development on the blot. Lane 1: untreated Jurkat cells, Lane 2: Jurkat cells kept in co-culture with JB6pcDNA5/FRT CAT, Lane 3: Jurkat cells kept in co-culture with JB6pcDNA5/FRT CAT prestimulated with concanavaline A, Lane 4: Jurkat cells kept in co-culture with JB6pcDNA5/FRT TRAIL, Lane 5: Jurkat cells kept in co-culture with JB6pcDNA5/FRT TRAIL prestimulated with concanavaline A.

    Journal: Annals of Surgical Innovation and Research

    Article Title: Expression of TNF-related apoptosis-inducing ligand (TRAIL) in keratinocytes mediates apoptotic cell death in allogenic T cells

    doi: 10.1186/1750-1164-3-13

    Figure Lengend Snippet: The effect of TRAIL expression in JB6-pcDNA5/FRT TRAIL cells on co-cultured Jurkat cells . A Phase-contrast-imaging of apoptotic Jurkat cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. b Annexin V/Propidiumiodide dyed Jurkat cells after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. Membrane fluorescence is characteristic for apoptosis. c Phase-contrast-imaging of a Jurkat cell after 48 hours of co-culture with JB6-pcDNA5/FRT CAT. d Annexin V/Propidiumiodide dyed Jurkat cell after 48 hours of co-culture with JB6-pcDNA5/FRT CAT. No membrane fluorescence could be detected. e Western blot stained with anti-Caspase 8 followed by alkaline phosphatase-conjugated secondary antibody and NBT/BCIP colour development on the blot. Lane 1: untreated Jurkat cells, Lane 2: Jurkat cells kept in co-culture with JB6pcDNA5/FRT CAT, Lane 3: Jurkat cells kept in co-culture with JB6pcDNA5/FRT CAT prestimulated with concanavaline A, Lane 4: Jurkat cells kept in co-culture with JB6pcDNA5/FRT TRAIL, Lane 5: Jurkat cells kept in co-culture with JB6pcDNA5/FRT TRAIL prestimulated with concanavaline A.

    Article Snippet: Generation of TRAIL producing cell clones Full length TRAIL cDNA was cloned from a human lymphoma cDNA library (Lym12, Invitrogen, CA) using -ATG GCT ATG ATG GAG GTC- as 5' and -TTA GCC AAC TAA AAA GGC- as 3' primer, according to the published sequence (GeneBank accession number: U37518 ), and cloned into the expression vector pcDNA5/FRT (Invitrogen).

    Techniques: Expressing, Cell Culture, Imaging, Co-Culture Assay, Fluorescence, Western Blot, Staining

    Co-culturing of JB6pcDNA5/FRT TRAIL with native T cells . a Phase-contrast-imaging of a apoptotic CD4 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. b Annexin V/Propidiumiodide dyed CD4 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. Membrane fluorescence is characteristic for apoptosis. c Phase-contrast-imaging of a CD8 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. d Annexin V/Propidiumiodide dyed CD8 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL.

    Journal: Annals of Surgical Innovation and Research

    Article Title: Expression of TNF-related apoptosis-inducing ligand (TRAIL) in keratinocytes mediates apoptotic cell death in allogenic T cells

    doi: 10.1186/1750-1164-3-13

    Figure Lengend Snippet: Co-culturing of JB6pcDNA5/FRT TRAIL with native T cells . a Phase-contrast-imaging of a apoptotic CD4 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. b Annexin V/Propidiumiodide dyed CD4 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. Membrane fluorescence is characteristic for apoptosis. c Phase-contrast-imaging of a CD8 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL. d Annexin V/Propidiumiodide dyed CD8 + cell after 48 hours of co-culture with JB6-pcDNA5/FRT TRAIL.

    Article Snippet: Generation of TRAIL producing cell clones Full length TRAIL cDNA was cloned from a human lymphoma cDNA library (Lym12, Invitrogen, CA) using -ATG GCT ATG ATG GAG GTC- as 5' and -TTA GCC AAC TAA AAA GGC- as 3' primer, according to the published sequence (GeneBank accession number: U37518 ), and cloned into the expression vector pcDNA5/FRT (Invitrogen).

    Techniques: Imaging, Co-Culture Assay, Fluorescence

    CUL5-NEDD8 conjugation is required for 17-AAG–induced client degradation but not for loss of client activity. HT29 human colon cancer cells were transfected with pcDNA5/FRT/TO plasmids containing HA-tagged CUL5 (CUL5-WT), an HA-tagged CUL5 triple

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

    Article Title: E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

    doi: 10.1073/pnas.1322412111

    Figure Lengend Snippet: CUL5-NEDD8 conjugation is required for 17-AAG–induced client degradation but not for loss of client activity. HT29 human colon cancer cells were transfected with pcDNA5/FRT/TO plasmids containing HA-tagged CUL5 (CUL5-WT), an HA-tagged CUL5 triple

    Article Snippet: Oligonucleotides were transfected into HT29 cells using DharmaFECT-4 (Dharmacon), BT474 cells using DharmaFECT-2, or HCT116 and H1975 cells using HiPerFect (Qiagen) and 25 nM siRNA according to manufacturer’s instructions. pcDNA5/FRT/TO plasmids (Invitrogen) containing HA-EV, HA-CUL5-WT, and HA-CUL5-∆NEDD8 were provided by Arno Alpi ( ) and transfected into cells using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions 24 h before siRNA transfection.

    Techniques: Conjugation Assay, Activity Assay, Transfection

    Depletion of Wdr82 decreases Setd1A occupancy and histone H3-Lys4 trimethylation near the transcription start site. A. Diagram of the inducible Wdr82 miRNA construct. Two 118-nt modified miR-30 hairpins for Wdr82 were tandemly subcloned into the pcDNA5/TO vector, which contains a C-terminal green fluorescent protein cDNA and is expressed under control of a tetracycline operator. Restriction enzyme sites used for subcloning are underlined. B. Inducible T-REx HEK293 cell lines that express tandem miRNA of human Wdr82, tandem scramble miRNA, or vector only were induced for 5 days with 1 μg/ml doxycycline. Nuclear extracts were prepared and analyzed by Western blotting using the indicated antisera. The results are shown for two independent clones that express the tandem miRNA of human Wdr82. C. Cell lines that express the tandem miRNA of Wdr82 were induced for 5 days with 1 μg/ml doxycycline. Histone H3-Lys4 methylation near the transcription start site of the PPIA , PABPC1 , and GAPDH genes and the promoter region of the HOXC8 gene were analyzed by ChIP and real-time PCR. The level of histone H3-Lys4 methylation in uninduced cells was set at 100%. The data represent a summary of three independent experiments. Error bars indicate the mean standard deviations, and P values were determined by a standard t test. Asterisks indicate P values of

    Journal: Molecular and Cellular Biology

    Article Title: Wdr82 Is a C-Terminal Domain-Binding Protein That Recruits the Setd1A Histone H3-Lys4 Methyltransferase Complex to Transcription Start Sites of Transcribed Human Genes ▿

    doi: 10.1128/MCB.01356-07

    Figure Lengend Snippet: Depletion of Wdr82 decreases Setd1A occupancy and histone H3-Lys4 trimethylation near the transcription start site. A. Diagram of the inducible Wdr82 miRNA construct. Two 118-nt modified miR-30 hairpins for Wdr82 were tandemly subcloned into the pcDNA5/TO vector, which contains a C-terminal green fluorescent protein cDNA and is expressed under control of a tetracycline operator. Restriction enzyme sites used for subcloning are underlined. B. Inducible T-REx HEK293 cell lines that express tandem miRNA of human Wdr82, tandem scramble miRNA, or vector only were induced for 5 days with 1 μg/ml doxycycline. Nuclear extracts were prepared and analyzed by Western blotting using the indicated antisera. The results are shown for two independent clones that express the tandem miRNA of human Wdr82. C. Cell lines that express the tandem miRNA of Wdr82 were induced for 5 days with 1 μg/ml doxycycline. Histone H3-Lys4 methylation near the transcription start site of the PPIA , PABPC1 , and GAPDH genes and the promoter region of the HOXC8 gene were analyzed by ChIP and real-time PCR. The level of histone H3-Lys4 methylation in uninduced cells was set at 100%. The data represent a summary of three independent experiments. Error bars indicate the mean standard deviations, and P values were determined by a standard t test. Asterisks indicate P values of

    Article Snippet: Expression vectors carrying human Setd1A or Wdr82 cDNAs were prepared using the pcDNA5/TO vector (Invitrogen), which carries an amino-terminal FLAG epitope, as previously described ( ).

    Techniques: Construct, Modification, Plasmid Preparation, Subcloning, Western Blot, Clone Assay, Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction