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    Stratagene quikchange site directed mutagenesis kit
    FOXO3a represses and FOXM1 induces the transcriptional activity of the human VEGF gene through a FHRE consensus site proximal to the transcription start site A) Effect of expression of FOXO3a and FOXM1 on VEGF promoter activity. Schematic representation of the VEGF-luciferase reporter construct, showing the consensus FHRE sequences. A 1741 bp VEGF promoter construct (positions −1,926 to −186 relative to the predominant 5′-transcription start site) was cloned into the XhoI and HindIII sites of the pGL3 basic vector (Promega, Southampton, United Kingdom). Putative forkhead site mutagenesis was performed using a Stratagene <t>QuikChange</t> site-directed mutagenesis kit with the oligonucleotides: Site1 (−178) (5′-ATCCCTCTTCTTTTTTCTTGGGCATTTTTTTTTAAAACTGTATTGT-3′), and Site2 (−319) (5′- TTGCTCTACTTCCCCGGGTCACTGTGGATTTTGGGGGCCAGCAGA-3′). B) MCF-7 cells were transiently transfected with 20 ng of either the wild-type, (VEGF pro-WT), mutant FHRE1 (VEGF pro-mut1), or mutant FHRE2 (VEGF pro-mut2) VEGF promoter/reporter and 0, 5, 10 or 20 ng of either the constitutively active FOXO3a(A3) or FOXM1(ΔN) expression vector. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. C) MDA-MB-231-FOXO3a(A3):ER and MDA-MB-231 cells were treated with 200 nmol/L 4-OHT for the indicated times. Nuclear extracts prepared were incubated with biotinylated wild-type or mutant FHRE2 oligonucleotides in the presence or absence of 5x molar excess of non-biotinylated wild-type or mutant FHRE2 oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using specific antibodies as indicated. D) The nuclear and cytoplasmic extracts prepared from BT474 cells treated with lapatinib for 0, 2 and 4 h were western blotted for proteins indicated (right panel). The nuclear extracts from the lapatinib-treated cells were also examined by pull-down assays using biotinylated wild-type or mutant FHRE2 oligonucleotides as described above. E) Chromatin immunoprecipitation (ChIP) analysis of the human VEGF promoter. MDA-MB-231-FOXO3a(A3):ER, MDA-MB-231 and BT474 cells described above were used for ChIP assays using IgG, anti-FOXO3a and anti-FOXM1 antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the VEGF FHRE2 containing region (−351/−186) and resolved in 2% agarose gel. Representative data from three independent experiments are shown.
    Quikchange Site Directed Mutagenesis Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quikchange site directed mutagenesis kit/product/Stratagene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    quikchange site directed mutagenesis kit - by Bioz Stars, 2021-06
    86/100 stars
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    86
    Stratagene site directed mutagenesis
    FOXO3a represses and FOXM1 induces the transcriptional activity of the human VEGF gene through a FHRE consensus site proximal to the transcription start site A) Effect of expression of FOXO3a and FOXM1 on VEGF promoter activity. Schematic representation of the VEGF-luciferase reporter construct, showing the consensus FHRE sequences. A 1741 bp VEGF promoter construct (positions −1,926 to −186 relative to the predominant 5′-transcription start site) was cloned into the XhoI and HindIII sites of the pGL3 basic vector (Promega, Southampton, United Kingdom). Putative forkhead site mutagenesis was performed using a Stratagene <t>QuikChange</t> site-directed mutagenesis kit with the oligonucleotides: Site1 (−178) (5′-ATCCCTCTTCTTTTTTCTTGGGCATTTTTTTTTAAAACTGTATTGT-3′), and Site2 (−319) (5′- TTGCTCTACTTCCCCGGGTCACTGTGGATTTTGGGGGCCAGCAGA-3′). B) MCF-7 cells were transiently transfected with 20 ng of either the wild-type, (VEGF pro-WT), mutant FHRE1 (VEGF pro-mut1), or mutant FHRE2 (VEGF pro-mut2) VEGF promoter/reporter and 0, 5, 10 or 20 ng of either the constitutively active FOXO3a(A3) or FOXM1(ΔN) expression vector. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. C) MDA-MB-231-FOXO3a(A3):ER and MDA-MB-231 cells were treated with 200 nmol/L 4-OHT for the indicated times. Nuclear extracts prepared were incubated with biotinylated wild-type or mutant FHRE2 oligonucleotides in the presence or absence of 5x molar excess of non-biotinylated wild-type or mutant FHRE2 oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using specific antibodies as indicated. D) The nuclear and cytoplasmic extracts prepared from BT474 cells treated with lapatinib for 0, 2 and 4 h were western blotted for proteins indicated (right panel). The nuclear extracts from the lapatinib-treated cells were also examined by pull-down assays using biotinylated wild-type or mutant FHRE2 oligonucleotides as described above. E) Chromatin immunoprecipitation (ChIP) analysis of the human VEGF promoter. MDA-MB-231-FOXO3a(A3):ER, MDA-MB-231 and BT474 cells described above were used for ChIP assays using IgG, anti-FOXO3a and anti-FOXM1 antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the VEGF FHRE2 containing region (−351/−186) and resolved in 2% agarose gel. Representative data from three independent experiments are shown.
    Site Directed Mutagenesis, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/site directed mutagenesis/product/Stratagene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    site directed mutagenesis - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    86
    Stratagene quickchange site directed mutagenesis kit
    FOXO3a represses and FOXM1 induces the transcriptional activity of the human VEGF gene through a FHRE consensus site proximal to the transcription start site A) Effect of expression of FOXO3a and FOXM1 on VEGF promoter activity. Schematic representation of the VEGF-luciferase reporter construct, showing the consensus FHRE sequences. A 1741 bp VEGF promoter construct (positions −1,926 to −186 relative to the predominant 5′-transcription start site) was cloned into the XhoI and HindIII sites of the pGL3 basic vector (Promega, Southampton, United Kingdom). Putative forkhead site mutagenesis was performed using a Stratagene <t>QuikChange</t> site-directed mutagenesis kit with the oligonucleotides: Site1 (−178) (5′-ATCCCTCTTCTTTTTTCTTGGGCATTTTTTTTTAAAACTGTATTGT-3′), and Site2 (−319) (5′- TTGCTCTACTTCCCCGGGTCACTGTGGATTTTGGGGGCCAGCAGA-3′). B) MCF-7 cells were transiently transfected with 20 ng of either the wild-type, (VEGF pro-WT), mutant FHRE1 (VEGF pro-mut1), or mutant FHRE2 (VEGF pro-mut2) VEGF promoter/reporter and 0, 5, 10 or 20 ng of either the constitutively active FOXO3a(A3) or FOXM1(ΔN) expression vector. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. C) MDA-MB-231-FOXO3a(A3):ER and MDA-MB-231 cells were treated with 200 nmol/L 4-OHT for the indicated times. Nuclear extracts prepared were incubated with biotinylated wild-type or mutant FHRE2 oligonucleotides in the presence or absence of 5x molar excess of non-biotinylated wild-type or mutant FHRE2 oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using specific antibodies as indicated. D) The nuclear and cytoplasmic extracts prepared from BT474 cells treated with lapatinib for 0, 2 and 4 h were western blotted for proteins indicated (right panel). The nuclear extracts from the lapatinib-treated cells were also examined by pull-down assays using biotinylated wild-type or mutant FHRE2 oligonucleotides as described above. E) Chromatin immunoprecipitation (ChIP) analysis of the human VEGF promoter. MDA-MB-231-FOXO3a(A3):ER, MDA-MB-231 and BT474 cells described above were used for ChIP assays using IgG, anti-FOXO3a and anti-FOXM1 antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the VEGF FHRE2 containing region (−351/−186) and resolved in 2% agarose gel. Representative data from three independent experiments are shown.
    Quickchange Site Directed Mutagenesis Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quickchange site directed mutagenesis kit/product/Stratagene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    quickchange site directed mutagenesis kit - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    86
    Agilent technologies quikchange site directed mutagenesis kit
    FOXO3a represses and FOXM1 induces the transcriptional activity of the human VEGF gene through a FHRE consensus site proximal to the transcription start site A) Effect of expression of FOXO3a and FOXM1 on VEGF promoter activity. Schematic representation of the VEGF-luciferase reporter construct, showing the consensus FHRE sequences. A 1741 bp VEGF promoter construct (positions −1,926 to −186 relative to the predominant 5′-transcription start site) was cloned into the XhoI and HindIII sites of the pGL3 basic vector (Promega, Southampton, United Kingdom). Putative forkhead site mutagenesis was performed using a Stratagene <t>QuikChange</t> site-directed mutagenesis kit with the oligonucleotides: Site1 (−178) (5′-ATCCCTCTTCTTTTTTCTTGGGCATTTTTTTTTAAAACTGTATTGT-3′), and Site2 (−319) (5′- TTGCTCTACTTCCCCGGGTCACTGTGGATTTTGGGGGCCAGCAGA-3′). B) MCF-7 cells were transiently transfected with 20 ng of either the wild-type, (VEGF pro-WT), mutant FHRE1 (VEGF pro-mut1), or mutant FHRE2 (VEGF pro-mut2) VEGF promoter/reporter and 0, 5, 10 or 20 ng of either the constitutively active FOXO3a(A3) or FOXM1(ΔN) expression vector. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. C) MDA-MB-231-FOXO3a(A3):ER and MDA-MB-231 cells were treated with 200 nmol/L 4-OHT for the indicated times. Nuclear extracts prepared were incubated with biotinylated wild-type or mutant FHRE2 oligonucleotides in the presence or absence of 5x molar excess of non-biotinylated wild-type or mutant FHRE2 oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using specific antibodies as indicated. D) The nuclear and cytoplasmic extracts prepared from BT474 cells treated with lapatinib for 0, 2 and 4 h were western blotted for proteins indicated (right panel). The nuclear extracts from the lapatinib-treated cells were also examined by pull-down assays using biotinylated wild-type or mutant FHRE2 oligonucleotides as described above. E) Chromatin immunoprecipitation (ChIP) analysis of the human VEGF promoter. MDA-MB-231-FOXO3a(A3):ER, MDA-MB-231 and BT474 cells described above were used for ChIP assays using IgG, anti-FOXO3a and anti-FOXM1 antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the VEGF FHRE2 containing region (−351/−186) and resolved in 2% agarose gel. Representative data from three independent experiments are shown.
    Quikchange Site Directed Mutagenesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quikchange site directed mutagenesis kit/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    quikchange site directed mutagenesis kit - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    Image Search Results


    FOXO3a represses and FOXM1 induces the transcriptional activity of the human VEGF gene through a FHRE consensus site proximal to the transcription start site A) Effect of expression of FOXO3a and FOXM1 on VEGF promoter activity. Schematic representation of the VEGF-luciferase reporter construct, showing the consensus FHRE sequences. A 1741 bp VEGF promoter construct (positions −1,926 to −186 relative to the predominant 5′-transcription start site) was cloned into the XhoI and HindIII sites of the pGL3 basic vector (Promega, Southampton, United Kingdom). Putative forkhead site mutagenesis was performed using a Stratagene QuikChange site-directed mutagenesis kit with the oligonucleotides: Site1 (−178) (5′-ATCCCTCTTCTTTTTTCTTGGGCATTTTTTTTTAAAACTGTATTGT-3′), and Site2 (−319) (5′- TTGCTCTACTTCCCCGGGTCACTGTGGATTTTGGGGGCCAGCAGA-3′). B) MCF-7 cells were transiently transfected with 20 ng of either the wild-type, (VEGF pro-WT), mutant FHRE1 (VEGF pro-mut1), or mutant FHRE2 (VEGF pro-mut2) VEGF promoter/reporter and 0, 5, 10 or 20 ng of either the constitutively active FOXO3a(A3) or FOXM1(ΔN) expression vector. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. C) MDA-MB-231-FOXO3a(A3):ER and MDA-MB-231 cells were treated with 200 nmol/L 4-OHT for the indicated times. Nuclear extracts prepared were incubated with biotinylated wild-type or mutant FHRE2 oligonucleotides in the presence or absence of 5x molar excess of non-biotinylated wild-type or mutant FHRE2 oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using specific antibodies as indicated. D) The nuclear and cytoplasmic extracts prepared from BT474 cells treated with lapatinib for 0, 2 and 4 h were western blotted for proteins indicated (right panel). The nuclear extracts from the lapatinib-treated cells were also examined by pull-down assays using biotinylated wild-type or mutant FHRE2 oligonucleotides as described above. E) Chromatin immunoprecipitation (ChIP) analysis of the human VEGF promoter. MDA-MB-231-FOXO3a(A3):ER, MDA-MB-231 and BT474 cells described above were used for ChIP assays using IgG, anti-FOXO3a and anti-FOXM1 antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the VEGF FHRE2 containing region (−351/−186) and resolved in 2% agarose gel. Representative data from three independent experiments are shown.

    Journal: Oncogene

    Article Title: FOXO3a represses VEGF expression through FOXM1-dependent and -independent mechanisms in breast cancer

    doi: 10.1038/onc.2011.368

    Figure Lengend Snippet: FOXO3a represses and FOXM1 induces the transcriptional activity of the human VEGF gene through a FHRE consensus site proximal to the transcription start site A) Effect of expression of FOXO3a and FOXM1 on VEGF promoter activity. Schematic representation of the VEGF-luciferase reporter construct, showing the consensus FHRE sequences. A 1741 bp VEGF promoter construct (positions −1,926 to −186 relative to the predominant 5′-transcription start site) was cloned into the XhoI and HindIII sites of the pGL3 basic vector (Promega, Southampton, United Kingdom). Putative forkhead site mutagenesis was performed using a Stratagene QuikChange site-directed mutagenesis kit with the oligonucleotides: Site1 (−178) (5′-ATCCCTCTTCTTTTTTCTTGGGCATTTTTTTTTAAAACTGTATTGT-3′), and Site2 (−319) (5′- TTGCTCTACTTCCCCGGGTCACTGTGGATTTTGGGGGCCAGCAGA-3′). B) MCF-7 cells were transiently transfected with 20 ng of either the wild-type, (VEGF pro-WT), mutant FHRE1 (VEGF pro-mut1), or mutant FHRE2 (VEGF pro-mut2) VEGF promoter/reporter and 0, 5, 10 or 20 ng of either the constitutively active FOXO3a(A3) or FOXM1(ΔN) expression vector. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. C) MDA-MB-231-FOXO3a(A3):ER and MDA-MB-231 cells were treated with 200 nmol/L 4-OHT for the indicated times. Nuclear extracts prepared were incubated with biotinylated wild-type or mutant FHRE2 oligonucleotides in the presence or absence of 5x molar excess of non-biotinylated wild-type or mutant FHRE2 oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using specific antibodies as indicated. D) The nuclear and cytoplasmic extracts prepared from BT474 cells treated with lapatinib for 0, 2 and 4 h were western blotted for proteins indicated (right panel). The nuclear extracts from the lapatinib-treated cells were also examined by pull-down assays using biotinylated wild-type or mutant FHRE2 oligonucleotides as described above. E) Chromatin immunoprecipitation (ChIP) analysis of the human VEGF promoter. MDA-MB-231-FOXO3a(A3):ER, MDA-MB-231 and BT474 cells described above were used for ChIP assays using IgG, anti-FOXO3a and anti-FOXM1 antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the VEGF FHRE2 containing region (−351/−186) and resolved in 2% agarose gel. Representative data from three independent experiments are shown.

    Article Snippet: Putative forkhead site mutagenesis was performed using a Stratagene QuikChange site-directed mutagenesis kit.

    Techniques: Activity Assay, Expressing, Luciferase, Construct, Clone Assay, Plasmid Preparation, Mutagenesis, Transfection, Multiple Displacement Amplification, Incubation, Binding Assay, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis