gc-rich pcr system Search Results


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  • 94
    Millipore gc rich pcr
    Gc Rich Pcr, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gc rich polymerase chain reaction pcr system
    <t>PCR</t> assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S <t>rRNA</t>
    Gc Rich Polymerase Chain Reaction Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gc rich pcr system
    <t>PCR</t> assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S <t>rRNA</t>
    Gc Rich Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa gc rich pcr system
    Analysis of the FGFR2 promoter. A , mapping the transcription start site of FGFR2 in C3H10T1/2. The experimentally determined transcription initiation site of the mouse FGFR2 promoter by 5′-RLM-RACE is denoted as +1. The <t>cDNA</t> sequences of KGFR and bek begin at position +17 and +37, respectively. B , DNase I hypersensitive site analysis of the FGFR2 promoter region. Top panel , nuclei from C3H10T1/2, C2C12, MC3T3, and primary osteoblasts were treated with increasing amounts of DNase I ( left to right , shown by triangles ). After DNA extraction and digestion with EcoR, they were hybridized with the probe shown in V. Bottom panel , the arrowhead shows bands (about 6.3 kb) due to DNase I cleavage. C , DNase accessibility of FGFR2 gene in C3H10T1/2 cells. Top panel , nuclei from C3H10T1/2 were harvested and treated with 50 units of DNase for 10 min at 25 °C. Then the genomic DNA was purified and quantitated relative to DNA I from undigested nuclei using the primers described in the bottom panel by quantitative <t>PCR</t> and listed as percent protected. Each bar represents the means ± S.D. of three independent experiments.
    Gc Rich Pcr System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gc rich pcr system kit
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Gc Rich Pcr System Kit, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche 1x gc rich pcr system
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    1x Gc Rich Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    epigenomics gc rich pcr system
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Gc Rich Pcr System, supplied by epigenomics, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gc rich pcr system enzyme mix
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Gc Rich Pcr System Enzyme Mix, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gc rich pcr system buffer
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Gc Rich Pcr System Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gc rich pcr amplification system
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Gc Rich Pcr Amplification System, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa lataq gc rich pcr system
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Lataq Gc Rich Pcr System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gc rich pcr
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Gc Rich Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech gc rich pcr kit
    Separate control of <t>Notch1</t> transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time <t>RT-PCR.</t> The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.
    Gc Rich Pcr Kit, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA

    Journal: Journal of Clinical Microbiology

    Article Title: Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

    doi: 10.1128/JCM.00441-12

    Figure Lengend Snippet: PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA

    Article Snippet: PCRs with specific primers (0.2 μM each) for the amplification of DNA fragments of MAP1485, MAP1487, MAP1738, MAP2325, and MAP16S rRNA genes were performed from 250 ng of genomic DNA using the GC-Rich PCR system (Roche, Mannheim, Germany) according to the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Amplification

    Analysis of the FGFR2 promoter. A , mapping the transcription start site of FGFR2 in C3H10T1/2. The experimentally determined transcription initiation site of the mouse FGFR2 promoter by 5′-RLM-RACE is denoted as +1. The cDNA sequences of KGFR and bek begin at position +17 and +37, respectively. B , DNase I hypersensitive site analysis of the FGFR2 promoter region. Top panel , nuclei from C3H10T1/2, C2C12, MC3T3, and primary osteoblasts were treated with increasing amounts of DNase I ( left to right , shown by triangles ). After DNA extraction and digestion with EcoR, they were hybridized with the probe shown in V. Bottom panel , the arrowhead shows bands (about 6.3 kb) due to DNase I cleavage. C , DNase accessibility of FGFR2 gene in C3H10T1/2 cells. Top panel , nuclei from C3H10T1/2 were harvested and treated with 50 units of DNase for 10 min at 25 °C. Then the genomic DNA was purified and quantitated relative to DNA I from undigested nuclei using the primers described in the bottom panel by quantitative PCR and listed as percent protected. Each bar represents the means ± S.D. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells *Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells * S⃞

    doi: 10.1074/jbc.M808992200

    Figure Lengend Snippet: Analysis of the FGFR2 promoter. A , mapping the transcription start site of FGFR2 in C3H10T1/2. The experimentally determined transcription initiation site of the mouse FGFR2 promoter by 5′-RLM-RACE is denoted as +1. The cDNA sequences of KGFR and bek begin at position +17 and +37, respectively. B , DNase I hypersensitive site analysis of the FGFR2 promoter region. Top panel , nuclei from C3H10T1/2, C2C12, MC3T3, and primary osteoblasts were treated with increasing amounts of DNase I ( left to right , shown by triangles ). After DNA extraction and digestion with EcoR, they were hybridized with the probe shown in V. Bottom panel , the arrowhead shows bands (about 6.3 kb) due to DNase I cleavage. C , DNase accessibility of FGFR2 gene in C3H10T1/2 cells. Top panel , nuclei from C3H10T1/2 were harvested and treated with 50 units of DNase for 10 min at 25 °C. Then the genomic DNA was purified and quantitated relative to DNA I from undigested nuclei using the primers described in the bottom panel by quantitative PCR and listed as percent protected. Each bar represents the means ± S.D. of three independent experiments.

    Article Snippet: The 5′ cDNA end was amplified by PCR using the GC-Rich PCR system (Takara).

    Techniques: DNA Extraction, Purification, Real-time Polymerase Chain Reaction

    Separate control of Notch1 transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time RT-PCR. The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Separate control of Notch1 transcription by Klf4/Sp3 and p53. (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time RT-PCR. The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Transfection, Infection, Expressing, Quantitative RT-PCR, Plasmid Preparation

    Binding of endogenous Klf4 and Sp1/Sp3 proteins to the Notch1 promoter. (A) Predicted Klf4- and Sp1/Sp3 binding sites in the −340/−300 bp Notch1 promoter region. Shown is the nucleotide sequence of this region with, on top, the predicted binding sites for Klf4- and Sp1/Sp3. (B and C) Primary keratinocytes (HKC) and HeLa cells were processed for chromatin immunoprecipitation (ChIP) analysis with antibodies against Sp1, Sp3 (B), Klf4 (C) or Maz, as indicated, followed by amplification of two Notch1 promoter regions located between bp −740/−262 (Chip 1) and around −6600 bp (Chip 2), which contain and lack, respectively, putative Sp1/Sp3 and Klf4 binding sites. Amplification of the proximal promoter region of the p21 WAF1/Cip1 gene (Chip p21) was used as positive control. Un-precipitated chromatin preparations were used for parallel amplification reactions as ‘input’ DNA controls. (D) Chip assays with anti-Sp3 and Klf4 antibodies and non immune IgGs as in the previous panels were followed by real time PCR amplification of region1 of the Notch1 promoter. The amount of precipitated DNA was calculated relative to the total input chromatin and expressed as a percentage of the total according to the following formula [54] : percentage total = 2ΔCt×5, where ΔCt = Ct (input) − Ct (immunoprecipitation), and Ct is the cycle threshold.

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Binding of endogenous Klf4 and Sp1/Sp3 proteins to the Notch1 promoter. (A) Predicted Klf4- and Sp1/Sp3 binding sites in the −340/−300 bp Notch1 promoter region. Shown is the nucleotide sequence of this region with, on top, the predicted binding sites for Klf4- and Sp1/Sp3. (B and C) Primary keratinocytes (HKC) and HeLa cells were processed for chromatin immunoprecipitation (ChIP) analysis with antibodies against Sp1, Sp3 (B), Klf4 (C) or Maz, as indicated, followed by amplification of two Notch1 promoter regions located between bp −740/−262 (Chip 1) and around −6600 bp (Chip 2), which contain and lack, respectively, putative Sp1/Sp3 and Klf4 binding sites. Amplification of the proximal promoter region of the p21 WAF1/Cip1 gene (Chip p21) was used as positive control. Un-precipitated chromatin preparations were used for parallel amplification reactions as ‘input’ DNA controls. (D) Chip assays with anti-Sp3 and Klf4 antibodies and non immune IgGs as in the previous panels were followed by real time PCR amplification of region1 of the Notch1 promoter. The amount of precipitated DNA was calculated relative to the total input chromatin and expressed as a percentage of the total according to the following formula [54] : percentage total = 2ΔCt×5, where ΔCt = Ct (input) − Ct (immunoprecipitation), and Ct is the cycle threshold.

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Amplification, Positive Control, Real-time Polymerase Chain Reaction, Immunoprecipitation

    Opposite control of PolII recruitment to the Notch1 promoter by p53 and Klf4. (A) HeLa cells were infected with a recombinant adenovirus expressing wild-type p53 (Adp53) or GFP control (AdGFP) and processed for ChIP assays with antibodies against RNA polymerase II (α-PolII) and non-immune IgGs as control. PCR amplification of the indicated regions of the Notch1 gene was performed, in parallel with similar amplification of the input chromatin DNA. Right panels: for quantification of the results, the chromatin immunoprecipitated material was also analyzed by real time PCR amplification of the indicated regions of the Notch1 promoter, in parallel with input chromatin DNA, followed by a calculation of binding according to the same formula utilized in Fig. 7D . (B) Primary keratinocytes (HKC) were infected with a retroviral vector over-expressing Klf4 (pMSKlf4) or empty vector control (Ctrl) for 48 hours followed by ChIP assays for PolII binding as in the previous panel, including quantification of the results by real time PCR (right panels).

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Opposite control of PolII recruitment to the Notch1 promoter by p53 and Klf4. (A) HeLa cells were infected with a recombinant adenovirus expressing wild-type p53 (Adp53) or GFP control (AdGFP) and processed for ChIP assays with antibodies against RNA polymerase II (α-PolII) and non-immune IgGs as control. PCR amplification of the indicated regions of the Notch1 gene was performed, in parallel with similar amplification of the input chromatin DNA. Right panels: for quantification of the results, the chromatin immunoprecipitated material was also analyzed by real time PCR amplification of the indicated regions of the Notch1 promoter, in parallel with input chromatin DNA, followed by a calculation of binding according to the same formula utilized in Fig. 7D . (B) Primary keratinocytes (HKC) were infected with a retroviral vector over-expressing Klf4 (pMSKlf4) or empty vector control (Ctrl) for 48 hours followed by ChIP assays for PolII binding as in the previous panel, including quantification of the results by real time PCR (right panels).

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Infection, Recombinant, Expressing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Plasmid Preparation

    Up-regulation of Notch1 gene expression by Klf4 and Sp3 knock down. (A) Primary keratinocytes were transfected with two sets of siRNAs for Klf4, Sp1 or Sp3 in parallel with scrambled siRNA controls for 48 hours, followed by expression analysis of the targeted genes by real time RT-PCR and immunoblotting (left and middle panels, respectively) The same RNA samples were also analyzed for levels of Notch1 expression (right panel). (B) Primary keratinocytes, were transfected as in the previous panel with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) (left columns) or with siRNAs for Klf4, Sp1 and Sp3 (right columns), followed by real time RT-PCR analysis of Notch1 expression. Shown is the calculated average of four different experiments using 36β4 and 18S RNA for internal normalization. (C) HeLa and SCC13 cells were transfected with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) followed by determination of Notch1 expression by real-time RT-PCR as in the previous panel. Primary keratinocytes and SCC13 cells were transfected with siRNAs against the indicated genes followed by immunoblot analysis of Notch1 protein expression with γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression.

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Up-regulation of Notch1 gene expression by Klf4 and Sp3 knock down. (A) Primary keratinocytes were transfected with two sets of siRNAs for Klf4, Sp1 or Sp3 in parallel with scrambled siRNA controls for 48 hours, followed by expression analysis of the targeted genes by real time RT-PCR and immunoblotting (left and middle panels, respectively) The same RNA samples were also analyzed for levels of Notch1 expression (right panel). (B) Primary keratinocytes, were transfected as in the previous panel with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) (left columns) or with siRNAs for Klf4, Sp1 and Sp3 (right columns), followed by real time RT-PCR analysis of Notch1 expression. Shown is the calculated average of four different experiments using 36β4 and 18S RNA for internal normalization. (C) HeLa and SCC13 cells were transfected with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) followed by determination of Notch1 expression by real-time RT-PCR as in the previous panel. Primary keratinocytes and SCC13 cells were transfected with siRNAs against the indicated genes followed by immunoblot analysis of Notch1 protein expression with γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression.

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Autoradiography

    Mapping of the minimal functional region of the Notch1 promoter in primary human keratinocytes versus HeLa cells. (A) Different fragments of the human Notch1 promoter with decreasing 5′ ends were cloned into a luciferase reporter plasmid, followed by transient transfection into primary human keratinocytes and HeLa cells (black and grey bars, respectively) together with a Renilla minimal reporter for normalization. Promoter activity was measured 48 hours after transfection. Relative promoter activity of the various reporters in HKC versus HeLa was also calculated (right Table). (B) Fragments of the human Notch1 promoter with partial or total deletion of the sequence from the TSS to the initiation codon (lacking nucleotides −159 to −1, and −262 to −1, respectively) were cloned into a luciferase reporter plasmid, followed by transient transfection/promoter activity assays in primary human keratinocytes and HeLa cells as in the previous panel. Relative promoter activity in HKC versus HeLa was also calculated. (C) Total RNA from primary keratinocytes and various cancer cells lines, including PC3, Caski, and HeLa from two different sources (HeLa#1 and 2), was utilized for RT-PCR amplification of the 5′ UTR region of the Notch 1 gene (primer position −262/−174). Note the faster migrating band obtained with the HeLa samples. Cloning and sequencing of the overlapping genomic region (from position −392 bp to −1 of the Notch1 promoter) showed a deletion from position −215 to −202 in HeLa cells. (D) The same Notch1 genomic region plus/minus the above deletion was cloned into a luciferase reporter plasmid, followed by promoter activity assays in HKC versus HeLa cells, measured as in (A) and (B).

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Mapping of the minimal functional region of the Notch1 promoter in primary human keratinocytes versus HeLa cells. (A) Different fragments of the human Notch1 promoter with decreasing 5′ ends were cloned into a luciferase reporter plasmid, followed by transient transfection into primary human keratinocytes and HeLa cells (black and grey bars, respectively) together with a Renilla minimal reporter for normalization. Promoter activity was measured 48 hours after transfection. Relative promoter activity of the various reporters in HKC versus HeLa was also calculated (right Table). (B) Fragments of the human Notch1 promoter with partial or total deletion of the sequence from the TSS to the initiation codon (lacking nucleotides −159 to −1, and −262 to −1, respectively) were cloned into a luciferase reporter plasmid, followed by transient transfection/promoter activity assays in primary human keratinocytes and HeLa cells as in the previous panel. Relative promoter activity in HKC versus HeLa was also calculated. (C) Total RNA from primary keratinocytes and various cancer cells lines, including PC3, Caski, and HeLa from two different sources (HeLa#1 and 2), was utilized for RT-PCR amplification of the 5′ UTR region of the Notch 1 gene (primer position −262/−174). Note the faster migrating band obtained with the HeLa samples. Cloning and sequencing of the overlapping genomic region (from position −392 bp to −1 of the Notch1 promoter) showed a deletion from position −215 to −202 in HeLa cells. (D) The same Notch1 genomic region plus/minus the above deletion was cloned into a luciferase reporter plasmid, followed by promoter activity assays in HKC versus HeLa cells, measured as in (A) and (B).

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Functional Assay, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification

    Transcription of various regions of the Notch1 gene in primary human keratinocytes versus keratinocyte-derived cancer cell lines. (A) Organization of the Notch1 gene, with an indication of the open reading frame (ORF) with coding exons and intervening introns (black thick boxes and lines, respectively) and 5′ and 3′ untranslated regions (UTR). The position of the different sets of primers utilized for real time RT-PCR analysis is also indicated (thin arrows). (B) Total RNA from primary human keratinocytes (HKC), cervical carcinoma cells (HeLa, Caski and SiHa), and skin (SCC13) and oral (SCCO28) squamous carcinoma cells was analyzed by real-time RT-PCR with primers corresponding to different region of the Notch1 gene as indicated in (A). For this and all other figures, values were normalized to 36B4 and/or 18S RNA levels, and expressed as relative to those in primary keratinocytes.

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Transcription of various regions of the Notch1 gene in primary human keratinocytes versus keratinocyte-derived cancer cell lines. (A) Organization of the Notch1 gene, with an indication of the open reading frame (ORF) with coding exons and intervening introns (black thick boxes and lines, respectively) and 5′ and 3′ untranslated regions (UTR). The position of the different sets of primers utilized for real time RT-PCR analysis is also indicated (thin arrows). (B) Total RNA from primary human keratinocytes (HKC), cervical carcinoma cells (HeLa, Caski and SiHa), and skin (SCC13) and oral (SCCO28) squamous carcinoma cells was analyzed by real-time RT-PCR with primers corresponding to different region of the Notch1 gene as indicated in (A). For this and all other figures, values were normalized to 36B4 and/or 18S RNA levels, and expressed as relative to those in primary keratinocytes.

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Derivative Assay, Quantitative RT-PCR

    Negative control of Notch1 gene expression by Klf4. (A) Primary keratinocytes were co-transfected with a reporter containing the minimal functional Notch1 promoter (from -392pGL4) together with an expression vector for human Klf4 or empty vector control. Renilla minimal reporter was used for internal normalization, and the promoter activity was measured 48 hours after transfection. Shown are the results of two different experiments. (B) Primary keratinocytes were infected with a retroviral vector expressing KlfF4 (pMSKlf4) or an empty vector control and harvested after 48 hours, followed by determination of Klf4 and Notch1 mRNA expression by real-time PCR. Efficiency of infection with the pMSKlf4 virus was also assessed by the widespread morphological changes with flattening of cells (upper panels). (C) Primary keratinocytes were infected with a KlfF4 expressing retrovirus versus an empty vector control as in the previous panel, followed by immunoblot analysis with antibodies against Notch1, Klf4 and γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression. Similar results were obtained in a second independent experiment.

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Negative control of Notch1 gene expression by Klf4. (A) Primary keratinocytes were co-transfected with a reporter containing the minimal functional Notch1 promoter (from -392pGL4) together with an expression vector for human Klf4 or empty vector control. Renilla minimal reporter was used for internal normalization, and the promoter activity was measured 48 hours after transfection. Shown are the results of two different experiments. (B) Primary keratinocytes were infected with a retroviral vector expressing KlfF4 (pMSKlf4) or an empty vector control and harvested after 48 hours, followed by determination of Klf4 and Notch1 mRNA expression by real-time PCR. Efficiency of infection with the pMSKlf4 virus was also assessed by the widespread morphological changes with flattening of cells (upper panels). (C) Primary keratinocytes were infected with a KlfF4 expressing retrovirus versus an empty vector control as in the previous panel, followed by immunoblot analysis with antibodies against Notch1, Klf4 and γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression. Similar results were obtained in a second independent experiment.

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Negative Control, Expressing, Transfection, Functional Assay, Plasmid Preparation, Activity Assay, Infection, Real-time Polymerase Chain Reaction, Autoradiography

    Methylation state of the Notch1 regulatory region and expression of various Sp/KLF family members in primary keratinocytes versus keratinocyte-derived cancer cell lines. (A) Nuclear extracts from primary human keratinocytes (HKC) and HeLa cells were immunoprecipitated with antibodies against methylated DNA, followed by PCR analysis of the precipitated DNA with primers specific for the indicated regions of the Notch1 promoter and first intronic region. PCR with primers specific for a known methylated gene was used as positive control. (B) Total RNA samples from primary human keratinocytes (HKC) and the indicated cancer cell lines were analyzed by real time RT-PCR with primers specific for Sp1, Sp3, Klf4, KLF5 and KLF10a. (C) Primary keratinocytes, HeLa and SCC13 cells were analyzed by immunoblotting with antibodies against Sp1, Sp3 and Klf4, with β-actin as equal loading control.

    Journal: PLoS ONE

    Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

    doi: 10.1371/journal.pone.0010369

    Figure Lengend Snippet: Methylation state of the Notch1 regulatory region and expression of various Sp/KLF family members in primary keratinocytes versus keratinocyte-derived cancer cell lines. (A) Nuclear extracts from primary human keratinocytes (HKC) and HeLa cells were immunoprecipitated with antibodies against methylated DNA, followed by PCR analysis of the precipitated DNA with primers specific for the indicated regions of the Notch1 promoter and first intronic region. PCR with primers specific for a known methylated gene was used as positive control. (B) Total RNA samples from primary human keratinocytes (HKC) and the indicated cancer cell lines were analyzed by real time RT-PCR with primers specific for Sp1, Sp3, Klf4, KLF5 and KLF10a. (C) Primary keratinocytes, HeLa and SCC13 cells were analyzed by immunoblotting with antibodies against Sp1, Sp3 and Klf4, with β-actin as equal loading control.

    Article Snippet: All PCRs for the GC-rich Notch1 promoter sequence were performed using GC-rich PCR kit system (Roche).

    Techniques: Methylation, Expressing, Derivative Assay, Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR

    CREB promotes insulin/IGF signaling via induction of IRS2. ( A ) Western blot analysis of IRS1 and IRS2 levels in MIN-6 cells treated with forskolin for increasing times in serum-supplemented medium. Cells were infected with A-CREB or control GFP adenovirus as indicated. ( B ) Transient transfection assay of 293T cells with IRS2 promoter construct or control somatostatin CRE luciferase plasmid. Cells treated with forskolin or DMSO vehicle. Cotransfection with A-CREB expression plasmid is indicated. ( C ) Chromatin immunoprecipitation assay of the IRS2 promoter using CREB 244 antiserum. Presence of the IRS2 promoter or negative control GAPDH in CREB immunoprecipitates determined by PCR amplification of anti-CREB or control IgG immunoprecipitates. ( D ) IRS2 expression is disrupted in A-CREB transgenic mice. ( Top ) Immunohistochemical staining of pancreatic sections from wild-type and RIP A-CREB transgenic mice using anti-IRS2 antiserum. ( Bottom ) Western blot assay of islet-cell extracts from wild-type and A-CREB mice using anti-IRS2 antiserum. Comparable levels of CREB protein in transgenic and wild-type lysates. ( E ) cAMP-dependent induction of IRS-2 potentiates growth factor signaling to Akt. Effect of IGF1 on Akt phosphorylation at Thr 308 in cells pretreated with forskolin or vehicle under serum-starved conditions for 8 h. Effect of A-CREB on IGF-stimulated Akt Thr 308 phosphorylation.

    Journal: Genes & Development

    Article Title: cAMP promotes pancreatic ?-cell survival via CREB-mediated induction of IRS2

    doi: 10.1101/gad.1097103

    Figure Lengend Snippet: CREB promotes insulin/IGF signaling via induction of IRS2. ( A ) Western blot analysis of IRS1 and IRS2 levels in MIN-6 cells treated with forskolin for increasing times in serum-supplemented medium. Cells were infected with A-CREB or control GFP adenovirus as indicated. ( B ) Transient transfection assay of 293T cells with IRS2 promoter construct or control somatostatin CRE luciferase plasmid. Cells treated with forskolin or DMSO vehicle. Cotransfection with A-CREB expression plasmid is indicated. ( C ) Chromatin immunoprecipitation assay of the IRS2 promoter using CREB 244 antiserum. Presence of the IRS2 promoter or negative control GAPDH in CREB immunoprecipitates determined by PCR amplification of anti-CREB or control IgG immunoprecipitates. ( D ) IRS2 expression is disrupted in A-CREB transgenic mice. ( Top ) Immunohistochemical staining of pancreatic sections from wild-type and RIP A-CREB transgenic mice using anti-IRS2 antiserum. ( Bottom ) Western blot assay of islet-cell extracts from wild-type and A-CREB mice using anti-IRS2 antiserum. Comparable levels of CREB protein in transgenic and wild-type lysates. ( E ) cAMP-dependent induction of IRS-2 potentiates growth factor signaling to Akt. Effect of IGF1 on Akt phosphorylation at Thr 308 in cells pretreated with forskolin or vehicle under serum-starved conditions for 8 h. Effect of A-CREB on IGF-stimulated Akt Thr 308 phosphorylation.

    Article Snippet: PCR was performed with primers complementary to the IRS2 promoter region (forward –1244/–1225, reverse –1092/–1106) or to the GAPDH gene (forward 586/605, reverse 1037/1018) as control, by using the GC-rich PCR Kit (Roche).

    Techniques: Western Blot, Infection, Transient Transfection Assay, Construct, Luciferase, Plasmid Preparation, Cotransfection, Expressing, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Amplification, Transgenic Assay, Mouse Assay, Immunohistochemistry, Staining