anti-pol ii Santa Cruz Biotechnology Search Results


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  • 95
    Millipore rna pol ii
    <t>RBPJ</t> and SRF cooperated to regulate gene expression in SMC. ( A ) Coordinately induced and repressed genes were categorized based upon the cluster analysis shown in Figure 2A . ( B ) Quantification of <t>RNA</t> Pol II binding centered at RBPJ summits in control, RBPJ and RBPJ/SRF double knockdown cell lines for the indicated cluster from Figure 2A . Genes induced (top) or repressed (bottom) by RBPJ knockdown were analyzed separately. ( C ) RNA Pol II binding to and mRNA expression of the indicated SMC-specific genes was measured by targeted ChIP and semi-quantitative RT assays, respectively, in RBPJ knockdown and RBPJ/SRF double knockdown cells. ( D ) UCSC browser screenshot highlighting changes in RNA Pol II binding and mRNA induction at the MICAL2 gene in RBPJ and RBPJ/SRF double knockdown Hu Ao SMC. A previously un-annotated transcription start was identified and is marked by an arrow.
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    Santa Cruz Biotechnology santa cruz antibodies pol ii
    HIF1 or HIF2 target gene promoters/enhancers are bound by distinct <t>HIF1α/Pol</t> II or HIF2α/Pol II transcriptional complexes and formation of these transcriptional complexes depends on STAT3 or <t>USF2</t> activity. Sonicated chromatin from normoxic RCC4, RCC4/STAT3 shRNA, RCC4/USF2 shRNA or hypoxic Hep3B cells or Hep3B/USF2 shRNA cell was subjected to anti-Pol II ChIP, the precipitated protein/DNA complexes were then subjected to a secondary ChIP using HIF1α or HIF2α antibodies. Precipitated DNA was analyzed for HIF target gene promoter and results were displayed as percent of relative fold of binding. A ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 (two left columns) and mRNA levels of CA9 and PGK1 in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). B ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO (two left columns) and mRNA levels of PAI-1 and EPO in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). C ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 and fold of induction of CA9 and PGK1 gene expression in hypoxic Hep3B and Hep3B/USF2 shRNA cells. D ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO and fold of induction of PAI1 and EPO in hypoxic Hep3B and Hep3B/USF2 shRNA cells.
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    Santa Cruz Biotechnology rna polymerase ii pol ii antibodies
    Transcription factor assembly on the AP1 consensus response element in intact cells. (A) AP1 protein binding to the AP1 response element. HeLa cells were transfected with the AP1-tk Luc reporter gene together with the pcDNA myc-His hRARα expression vector. DNA-IP assays were performed after a 1-h treatment with TPA and/or atRA with antibodies (Ab) against AP1 proteins. As a control, a reporter gene containing an inactivated AP1 site and displaying no activity upon TPA treatment was used (AP1mut-tk Luc). Immunoprecipitated DNA was analyzed by semiquantitative PCR (top panels, 28 PCR cycles) and quantified by real-time PCR. Results are expressed as femtograms of DNA and displayed in the bar graph (bottom panel). The input lanes show that equivalent amounts of plasmid DNA were used for each DNA-IP. (B) atRA prevents <t>RNA</t> pol II loading on the AP1-responsive promoter. DNA-IP assays were performed as in panel A with either a nonphosphorylated RNA pol II CTD monoclonal antibody (RNA pol IIA, clone 8WG16, BaBCo) or a polyclonal antibody against total (phosphorylated and nonphosphorylated) RNA pol II (RNA pol II, total, sc899; Santa <t>Cruz</t> Biotechnology). (C) RARα is constitutively tethered to the AP1 binding site. DNA-IP assays were performed after transfection of cells with either the AP1-tk Luc or the DR5-tk Luc reporter genes together with RAR or RAR and RXR expression vectors, respectively. Antibodies directed against total RNA pol II or the RARα C terminus were used, and immunoprecipitates were analyzed for the presence of RARE or AP1 response element sequences.
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    Santa Cruz Biotechnology anti pol ii n20
    Transcription factor assembly on the AP1 consensus response element in intact cells. (A) AP1 protein binding to the AP1 response element. HeLa cells were transfected with the AP1-tk Luc reporter gene together with the pcDNA myc-His hRARα expression vector. DNA-IP assays were performed after a 1-h treatment with TPA and/or atRA with antibodies (Ab) against AP1 proteins. As a control, a reporter gene containing an inactivated AP1 site and displaying no activity upon TPA treatment was used (AP1mut-tk Luc). Immunoprecipitated DNA was analyzed by semiquantitative PCR (top panels, 28 PCR cycles) and quantified by real-time PCR. Results are expressed as femtograms of DNA and displayed in the bar graph (bottom panel). The input lanes show that equivalent amounts of plasmid DNA were used for each DNA-IP. (B) atRA prevents <t>RNA</t> pol II loading on the AP1-responsive promoter. DNA-IP assays were performed as in panel A with either a nonphosphorylated RNA pol II CTD monoclonal antibody (RNA pol IIA, clone 8WG16, BaBCo) or a polyclonal antibody against total (phosphorylated and nonphosphorylated) RNA pol II (RNA pol II, total, sc899; Santa <t>Cruz</t> Biotechnology). (C) RARα is constitutively tethered to the AP1 binding site. DNA-IP assays were performed after transfection of cells with either the AP1-tk Luc or the DR5-tk Luc reporter genes together with RAR or RAR and RXR expression vectors, respectively. Antibodies directed against total RNA pol II or the RARα C terminus were used, and immunoprecipitates were analyzed for the presence of RARE or AP1 response element sequences.
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    Santa Cruz Biotechnology anti pol ii n20x
    Transcription factor assembly on the AP1 consensus response element in intact cells. (A) AP1 protein binding to the AP1 response element. HeLa cells were transfected with the AP1-tk Luc reporter gene together with the pcDNA myc-His hRARα expression vector. DNA-IP assays were performed after a 1-h treatment with TPA and/or atRA with antibodies (Ab) against AP1 proteins. As a control, a reporter gene containing an inactivated AP1 site and displaying no activity upon TPA treatment was used (AP1mut-tk Luc). Immunoprecipitated DNA was analyzed by semiquantitative PCR (top panels, 28 PCR cycles) and quantified by real-time PCR. Results are expressed as femtograms of DNA and displayed in the bar graph (bottom panel). The input lanes show that equivalent amounts of plasmid DNA were used for each DNA-IP. (B) atRA prevents <t>RNA</t> pol II loading on the AP1-responsive promoter. DNA-IP assays were performed as in panel A with either a nonphosphorylated RNA pol II CTD monoclonal antibody (RNA pol IIA, clone 8WG16, BaBCo) or a polyclonal antibody against total (phosphorylated and nonphosphorylated) RNA pol II (RNA pol II, total, sc899; Santa <t>Cruz</t> Biotechnology). (C) RARα is constitutively tethered to the AP1 binding site. DNA-IP assays were performed after transfection of cells with either the AP1-tk Luc or the DR5-tk Luc reporter genes together with RAR or RAR and RXR expression vectors, respectively. Antibodies directed against total RNA pol II or the RARα C terminus were used, and immunoprecipitates were analyzed for the presence of RARE or AP1 response element sequences.
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    Santa Cruz Biotechnology anti phospho pol ii
    Transcription factor assembly on the AP1 consensus response element in intact cells. (A) AP1 protein binding to the AP1 response element. HeLa cells were transfected with the AP1-tk Luc reporter gene together with the pcDNA myc-His hRARα expression vector. DNA-IP assays were performed after a 1-h treatment with TPA and/or atRA with antibodies (Ab) against AP1 proteins. As a control, a reporter gene containing an inactivated AP1 site and displaying no activity upon TPA treatment was used (AP1mut-tk Luc). Immunoprecipitated DNA was analyzed by semiquantitative PCR (top panels, 28 PCR cycles) and quantified by real-time PCR. Results are expressed as femtograms of DNA and displayed in the bar graph (bottom panel). The input lanes show that equivalent amounts of plasmid DNA were used for each DNA-IP. (B) atRA prevents <t>RNA</t> pol II loading on the AP1-responsive promoter. DNA-IP assays were performed as in panel A with either a nonphosphorylated RNA pol II CTD monoclonal antibody (RNA pol IIA, clone 8WG16, BaBCo) or a polyclonal antibody against total (phosphorylated and nonphosphorylated) RNA pol II (RNA pol II, total, sc899; Santa <t>Cruz</t> Biotechnology). (C) RARα is constitutively tethered to the AP1 binding site. DNA-IP assays were performed after transfection of cells with either the AP1-tk Luc or the DR5-tk Luc reporter genes together with RAR or RAR and RXR expression vectors, respectively. Antibodies directed against total RNA pol II or the RARα C terminus were used, and immunoprecipitates were analyzed for the presence of RARE or AP1 response element sequences.
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    Santa Cruz Biotechnology rna pol ii
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology anti pol ii sc 899
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology pol ii antibody ctd4h8
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology anti rna pol ii ab
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology goat anti pol ii
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology mouse anti pol ii
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology rabbit anti pol ii
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology anti pol ii antibody n 20
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology anti rna pol ii n20
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology anti pol ii sc 33754 antibodies
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
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    Santa Cruz Biotechnology rabbit polyclonal anti pol ii
    Analysis of eEF1γ depletion. A : Indirect immunofluorescence obtained with the anti-Tom20 rabbit <t>polyclonal</t> and the anti-Vimentin monoclonal antibody in HeLa cells treated with siRNA-Control or with siRNA-eEF1γ. On the right side a higher magnification image of eEF1γ-depleted HeLa cells focusing on the formation of mitochondrial dysfunctional clusters. B : Real time RT-PCR analysis of the eEF1γ (left) and Vimentin (right) mRNAs in HeLa cells (siRNA-Control and siRNA-eEF1γ). The gene expression ratio between eEF1γ and GAPDH and between Vimentin and GAPDH are shown as means ± SD from three independent experiments performed in triplicate. C : The global protein synthesis of HeLa cells, transfected with either siRNA-Control or siRNA-eEF1γ and supplemented with S 35 labelled methionin and cystein, was visualized by autoradiography of the total protein lysates blotted to nitrocellulose membrane. Then the same membrane was incubated with the anti-eEF1γ rabbit polyclonal and the anti-Vimentin mouse monoclonal antibodies. Anti-β-actin monoclonal antibody was used to normalise the amount of protein loaded on the gel. D : Induced carbonylation pattern (oxidation level) of HeLa cells: untreated, treated with either siRNA-Control or siRNA-eEF1γ. The total protein carbonylation pattern was visualised by western blot with the anti-DNP antibody. Depletion of eEF1γ by siRNA was monitored using the anti-eEF1γ rabbit polyclonal antibody. The levels of Tom20 protein were monitored using the anti-Tom20 polyclonal antibody. Anti-alpha-tubulin monoclonal antibody was used to normalise the amount of protein loaded on the gel. E : Representative florescence images of HeLa cells treated with either siRNA-Control or siRNA-eEF1γ (top). The cellular level of superoxide was visualized by the MitoSOX (red) mitochondrial Superoxide Indicator staining. Nuclei were stained with Hoechst (blue). Histogram reporting fold of increase of MitoSOX fluorescence of siRNA-eEF1γ treated cells (lane 2) versus siRNA-Control treated cells (lane 1), shown as means ± SD from three independent experiments (bottom).
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    Santa Cruz Biotechnology anti polymerase ii polii
    Analysis of eEF1γ depletion. A : Indirect immunofluorescence obtained with the anti-Tom20 rabbit <t>polyclonal</t> and the anti-Vimentin monoclonal antibody in HeLa cells treated with siRNA-Control or with siRNA-eEF1γ. On the right side a higher magnification image of eEF1γ-depleted HeLa cells focusing on the formation of mitochondrial dysfunctional clusters. B : Real time RT-PCR analysis of the eEF1γ (left) and Vimentin (right) mRNAs in HeLa cells (siRNA-Control and siRNA-eEF1γ). The gene expression ratio between eEF1γ and GAPDH and between Vimentin and GAPDH are shown as means ± SD from three independent experiments performed in triplicate. C : The global protein synthesis of HeLa cells, transfected with either siRNA-Control or siRNA-eEF1γ and supplemented with S 35 labelled methionin and cystein, was visualized by autoradiography of the total protein lysates blotted to nitrocellulose membrane. Then the same membrane was incubated with the anti-eEF1γ rabbit polyclonal and the anti-Vimentin mouse monoclonal antibodies. Anti-β-actin monoclonal antibody was used to normalise the amount of protein loaded on the gel. D : Induced carbonylation pattern (oxidation level) of HeLa cells: untreated, treated with either siRNA-Control or siRNA-eEF1γ. The total protein carbonylation pattern was visualised by western blot with the anti-DNP antibody. Depletion of eEF1γ by siRNA was monitored using the anti-eEF1γ rabbit polyclonal antibody. The levels of Tom20 protein were monitored using the anti-Tom20 polyclonal antibody. Anti-alpha-tubulin monoclonal antibody was used to normalise the amount of protein loaded on the gel. E : Representative florescence images of HeLa cells treated with either siRNA-Control or siRNA-eEF1γ (top). The cellular level of superoxide was visualized by the MitoSOX (red) mitochondrial Superoxide Indicator staining. Nuclei were stained with Hoechst (blue). Histogram reporting fold of increase of MitoSOX fluorescence of siRNA-eEF1γ treated cells (lane 2) versus siRNA-Control treated cells (lane 1), shown as means ± SD from three independent experiments (bottom).
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    Image Search Results


    RBPJ and SRF cooperated to regulate gene expression in SMC. ( A ) Coordinately induced and repressed genes were categorized based upon the cluster analysis shown in Figure 2A . ( B ) Quantification of RNA Pol II binding centered at RBPJ summits in control, RBPJ and RBPJ/SRF double knockdown cell lines for the indicated cluster from Figure 2A . Genes induced (top) or repressed (bottom) by RBPJ knockdown were analyzed separately. ( C ) RNA Pol II binding to and mRNA expression of the indicated SMC-specific genes was measured by targeted ChIP and semi-quantitative RT assays, respectively, in RBPJ knockdown and RBPJ/SRF double knockdown cells. ( D ) UCSC browser screenshot highlighting changes in RNA Pol II binding and mRNA induction at the MICAL2 gene in RBPJ and RBPJ/SRF double knockdown Hu Ao SMC. A previously un-annotated transcription start was identified and is marked by an arrow.

    Journal: Nucleic Acids Research

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    doi: 10.1093/nar/gky562

    Figure Lengend Snippet: RBPJ and SRF cooperated to regulate gene expression in SMC. ( A ) Coordinately induced and repressed genes were categorized based upon the cluster analysis shown in Figure 2A . ( B ) Quantification of RNA Pol II binding centered at RBPJ summits in control, RBPJ and RBPJ/SRF double knockdown cell lines for the indicated cluster from Figure 2A . Genes induced (top) or repressed (bottom) by RBPJ knockdown were analyzed separately. ( C ) RNA Pol II binding to and mRNA expression of the indicated SMC-specific genes was measured by targeted ChIP and semi-quantitative RT assays, respectively, in RBPJ knockdown and RBPJ/SRF double knockdown cells. ( D ) UCSC browser screenshot highlighting changes in RNA Pol II binding and mRNA induction at the MICAL2 gene in RBPJ and RBPJ/SRF double knockdown Hu Ao SMC. A previously un-annotated transcription start was identified and is marked by an arrow.

    Article Snippet: Antibodies were RBPJ (D10A4) from Cell Signaling, SRF(G-20) from Santa Cruz biotechnology, RNA Pol II (8WG16) from Millipore and H3K9ac from Abcam.

    Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation

    RBPJ depletion in HuAo SMC promoted a differentiation gene program. ( A ) RNA Pol II binding and RNA expression in control and RBPJ depleted cells genes were assessed by ChIP-seq and RNA-seq respectively. The RBPJ-depletion-induced change in RNA Pol II binding within the 2000 bp around RBPJ summits was plotted against the RBPJ-depletion-induced change in RNA levels for the genes closest to that specific RBPJ binding site. Genes with RNA level measurements of

    Journal: Nucleic Acids Research

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    doi: 10.1093/nar/gky562

    Figure Lengend Snippet: RBPJ depletion in HuAo SMC promoted a differentiation gene program. ( A ) RNA Pol II binding and RNA expression in control and RBPJ depleted cells genes were assessed by ChIP-seq and RNA-seq respectively. The RBPJ-depletion-induced change in RNA Pol II binding within the 2000 bp around RBPJ summits was plotted against the RBPJ-depletion-induced change in RNA levels for the genes closest to that specific RBPJ binding site. Genes with RNA level measurements of

    Article Snippet: Antibodies were RBPJ (D10A4) from Cell Signaling, SRF(G-20) from Santa Cruz biotechnology, RNA Pol II (8WG16) from Millipore and H3K9ac from Abcam.

    Techniques: Binding Assay, RNA Expression, Chromatin Immunoprecipitation, RNA Sequencing Assay

    HIF1 or HIF2 target gene promoters/enhancers are bound by distinct HIF1α/Pol II or HIF2α/Pol II transcriptional complexes and formation of these transcriptional complexes depends on STAT3 or USF2 activity. Sonicated chromatin from normoxic RCC4, RCC4/STAT3 shRNA, RCC4/USF2 shRNA or hypoxic Hep3B cells or Hep3B/USF2 shRNA cell was subjected to anti-Pol II ChIP, the precipitated protein/DNA complexes were then subjected to a secondary ChIP using HIF1α or HIF2α antibodies. Precipitated DNA was analyzed for HIF target gene promoter and results were displayed as percent of relative fold of binding. A ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 (two left columns) and mRNA levels of CA9 and PGK1 in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). B ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO (two left columns) and mRNA levels of PAI-1 and EPO in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). C ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 and fold of induction of CA9 and PGK1 gene expression in hypoxic Hep3B and Hep3B/USF2 shRNA cells. D ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO and fold of induction of PAI1 and EPO in hypoxic Hep3B and Hep3B/USF2 shRNA cells.

    Journal: PLoS ONE

    Article Title: STAT3 or USF2 Contributes to HIF Target Gene Specificity

    doi: 10.1371/journal.pone.0072358

    Figure Lengend Snippet: HIF1 or HIF2 target gene promoters/enhancers are bound by distinct HIF1α/Pol II or HIF2α/Pol II transcriptional complexes and formation of these transcriptional complexes depends on STAT3 or USF2 activity. Sonicated chromatin from normoxic RCC4, RCC4/STAT3 shRNA, RCC4/USF2 shRNA or hypoxic Hep3B cells or Hep3B/USF2 shRNA cell was subjected to anti-Pol II ChIP, the precipitated protein/DNA complexes were then subjected to a secondary ChIP using HIF1α or HIF2α antibodies. Precipitated DNA was analyzed for HIF target gene promoter and results were displayed as percent of relative fold of binding. A ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 (two left columns) and mRNA levels of CA9 and PGK1 in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). B ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO (two left columns) and mRNA levels of PAI-1 and EPO in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). C ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 and fold of induction of CA9 and PGK1 gene expression in hypoxic Hep3B and Hep3B/USF2 shRNA cells. D ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO and fold of induction of PAI1 and EPO in hypoxic Hep3B and Hep3B/USF2 shRNA cells.

    Article Snippet: Anti-HIF1α (NB 100-134B3, Novus), anti-HIF2α (NB 100-122, Novus), anti-USF2 (C-20, SC-862, Santa Cruz), anti-STAT3 (K-15, sc-483; Santa Cruz), and anti-POL II (H-224, SC-9001, Santa Cruz) antibodies were used for protein-DNA complex precipitation, whereas rabbit preimmune serum served as a background control.

    Techniques: Activity Assay, Sonication, shRNA, Chromatin Immunoprecipitation, Binding Assay, Expressing

    Transcription factor assembly on the AP1 consensus response element in intact cells. (A) AP1 protein binding to the AP1 response element. HeLa cells were transfected with the AP1-tk Luc reporter gene together with the pcDNA myc-His hRARα expression vector. DNA-IP assays were performed after a 1-h treatment with TPA and/or atRA with antibodies (Ab) against AP1 proteins. As a control, a reporter gene containing an inactivated AP1 site and displaying no activity upon TPA treatment was used (AP1mut-tk Luc). Immunoprecipitated DNA was analyzed by semiquantitative PCR (top panels, 28 PCR cycles) and quantified by real-time PCR. Results are expressed as femtograms of DNA and displayed in the bar graph (bottom panel). The input lanes show that equivalent amounts of plasmid DNA were used for each DNA-IP. (B) atRA prevents RNA pol II loading on the AP1-responsive promoter. DNA-IP assays were performed as in panel A with either a nonphosphorylated RNA pol II CTD monoclonal antibody (RNA pol IIA, clone 8WG16, BaBCo) or a polyclonal antibody against total (phosphorylated and nonphosphorylated) RNA pol II (RNA pol II, total, sc899; Santa Cruz Biotechnology). (C) RARα is constitutively tethered to the AP1 binding site. DNA-IP assays were performed after transfection of cells with either the AP1-tk Luc or the DR5-tk Luc reporter genes together with RAR or RAR and RXR expression vectors, respectively. Antibodies directed against total RNA pol II or the RARα C terminus were used, and immunoprecipitates were analyzed for the presence of RARE or AP1 response element sequences.

    Journal: Molecular and Cellular Biology

    Article Title: Retinoic Acid Receptors Inhibit AP1 Activation by Regulating Extracellular Signal-Regulated Kinase and CBP Recruitment to an AP1-Responsive Promoter

    doi: 10.1128/MCB.22.13.4522-4534.2002

    Figure Lengend Snippet: Transcription factor assembly on the AP1 consensus response element in intact cells. (A) AP1 protein binding to the AP1 response element. HeLa cells were transfected with the AP1-tk Luc reporter gene together with the pcDNA myc-His hRARα expression vector. DNA-IP assays were performed after a 1-h treatment with TPA and/or atRA with antibodies (Ab) against AP1 proteins. As a control, a reporter gene containing an inactivated AP1 site and displaying no activity upon TPA treatment was used (AP1mut-tk Luc). Immunoprecipitated DNA was analyzed by semiquantitative PCR (top panels, 28 PCR cycles) and quantified by real-time PCR. Results are expressed as femtograms of DNA and displayed in the bar graph (bottom panel). The input lanes show that equivalent amounts of plasmid DNA were used for each DNA-IP. (B) atRA prevents RNA pol II loading on the AP1-responsive promoter. DNA-IP assays were performed as in panel A with either a nonphosphorylated RNA pol II CTD monoclonal antibody (RNA pol IIA, clone 8WG16, BaBCo) or a polyclonal antibody against total (phosphorylated and nonphosphorylated) RNA pol II (RNA pol II, total, sc899; Santa Cruz Biotechnology). (C) RARα is constitutively tethered to the AP1 binding site. DNA-IP assays were performed after transfection of cells with either the AP1-tk Luc or the DR5-tk Luc reporter genes together with RAR or RAR and RXR expression vectors, respectively. Antibodies directed against total RNA pol II or the RARα C terminus were used, and immunoprecipitates were analyzed for the presence of RARE or AP1 response element sequences.

    Article Snippet: Anti-RNA polymerase II (pol II) antibodies were from Santa Cruz Biotechnology (total RNA pol II, sc 899) and Covance/BabCo (anti-RNA pol IIA, 8WG16), and the DRIP205 antiserum was a gift from C. Rachez and L. P. Freedman.

    Techniques: Protein Binding, Transfection, Expressing, Plasmid Preparation, Activity Assay, Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Binding Assay

    BET inhibitor JQ1 suppresses TOPBP1 and WEE1 expression ( A ) Venn diagram and significance of overlap for genes occupied by BRD4, decrease BRD4 binding and downregulated by JQ1 and implicated in DNA damage signaling and repair or cell cycle checkpoint. The overlap was used to identify JQ1-regulated direct BRD4 target genes. ( B ) List of direct BRD4 target genes implicated in DNA damage signaling and repair or cell cycle checkpoint regulated by JQ1. ( C ) BRD4 ChIP-seq and nascent RNA-seq normalized reads from control and JQ1-treated OVCAR3 cells were aligned for WEE1 and TOPBP1 genomic loci. ( D ) Validation of TOPBP1 and WEE1 downregulation by JQ1. Relative primary transcript expression level of the indicated genes was measured by qRT-PCR with or without 250 nM JQ1 treatment for 30 mins. n=3, * p

    Journal: Cell reports

    Article Title: BET bromodomain inhibition synergizes with PARP inhibitor in epithelial ovarian cancer

    doi: 10.1016/j.celrep.2017.11.095

    Figure Lengend Snippet: BET inhibitor JQ1 suppresses TOPBP1 and WEE1 expression ( A ) Venn diagram and significance of overlap for genes occupied by BRD4, decrease BRD4 binding and downregulated by JQ1 and implicated in DNA damage signaling and repair or cell cycle checkpoint. The overlap was used to identify JQ1-regulated direct BRD4 target genes. ( B ) List of direct BRD4 target genes implicated in DNA damage signaling and repair or cell cycle checkpoint regulated by JQ1. ( C ) BRD4 ChIP-seq and nascent RNA-seq normalized reads from control and JQ1-treated OVCAR3 cells were aligned for WEE1 and TOPBP1 genomic loci. ( D ) Validation of TOPBP1 and WEE1 downregulation by JQ1. Relative primary transcript expression level of the indicated genes was measured by qRT-PCR with or without 250 nM JQ1 treatment for 30 mins. n=3, * p

    Article Snippet: The following antibodies were used to perform ChIP: anti-BRD4 (Bethyl Laboratories), anti-RNA polymerase II (Santa Cruz).

    Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Quantitative RT-PCR

    A significant decrease in transcription factor C/EBPβ RNA levels, but not C/EBPα, TEF-1, and TEF-5, is detected in the placenta with maternal obesity. Levels of C/EBPα, C/EBPβ, TEF-1, and TEF-5 in total RNA from placental

    Journal: The Journal of Biological Chemistry

    Article Title: CCAAT-enhancer-binding Protein β (C/EBPβ) and Downstream Human Placental Growth Hormone Genes Are Targets for Dysregulation in Pregnancies Complicated by Maternal Obesity *

    doi: 10.1074/jbc.M113.474999

    Figure Lengend Snippet: A significant decrease in transcription factor C/EBPβ RNA levels, but not C/EBPα, TEF-1, and TEF-5, is detected in the placenta with maternal obesity. Levels of C/EBPα, C/EBPβ, TEF-1, and TEF-5 in total RNA from placental

    Article Snippet: Soluble chromatin was immunoprecipitated with 4 μg of antibodies against C/EBPβ (Santa Cruz) or RNA polymerase II (Santa Cruz, sc-899 and Covance, MMS-134R) overnight at 4 °C as well as with normal rabbit immunoglobulins (Millipore) as a control.

    Techniques:

    Knockdown of human C/EBPβ in JEG-3 cells results in a decrease in endogenous CS and GH-V RNA levels. JEG-3 cells were treated with siRNAs for C/EBPβ or a “scrambled sequence” as a negative control for 72 h. After siRNA

    Journal: The Journal of Biological Chemistry

    Article Title: CCAAT-enhancer-binding Protein β (C/EBPβ) and Downstream Human Placental Growth Hormone Genes Are Targets for Dysregulation in Pregnancies Complicated by Maternal Obesity *

    doi: 10.1074/jbc.M113.474999

    Figure Lengend Snippet: Knockdown of human C/EBPβ in JEG-3 cells results in a decrease in endogenous CS and GH-V RNA levels. JEG-3 cells were treated with siRNAs for C/EBPβ or a “scrambled sequence” as a negative control for 72 h. After siRNA

    Article Snippet: Soluble chromatin was immunoprecipitated with 4 μg of antibodies against C/EBPβ (Santa Cruz) or RNA polymerase II (Santa Cruz, sc-899 and Covance, MMS-134R) overnight at 4 °C as well as with normal rabbit immunoglobulins (Millipore) as a control.

    Techniques: Sequencing, Negative Control

    Transcription on the mouse β-globin LCR HSs in HMBA-treated MEL cells ( A ) RT-PCR was performed in MEL cells at the four stages before and after transcriptional induction with or without reverse transcriptase (RT + or RT − ) and analysed as described in Figure 1 (B). ( B ) ChIP was performed with antibodies specific to RNA polymerase II over a time course. Relative intensity was determined by quantitatively comparing immunoprecipitated DNA with input for the indicated amplicons and normalizing to the intensity at the actin gene. Normal rabbit IgG (IgG) served as experimental control. The results are average of 3–4 independent experiments ± S.E.M.

    Journal: Bioscience Reports

    Article Title: Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus

    doi: 10.1042/BSR20140126

    Figure Lengend Snippet: Transcription on the mouse β-globin LCR HSs in HMBA-treated MEL cells ( A ) RT-PCR was performed in MEL cells at the four stages before and after transcriptional induction with or without reverse transcriptase (RT + or RT − ) and analysed as described in Figure 1 (B). ( B ) ChIP was performed with antibodies specific to RNA polymerase II over a time course. Relative intensity was determined by quantitatively comparing immunoprecipitated DNA with input for the indicated amplicons and normalizing to the intensity at the actin gene. Normal rabbit IgG (IgG) served as experimental control. The results are average of 3–4 independent experiments ± S.E.M.

    Article Snippet: Chromatin was fragmented by MNase digestion and sonication into mainly mononucleosomes and then was incubated with RNA polymerase II antibodies (Santa Cruz Biotechnology) or normal rabbit IgG (Santa Cruz Biotechnology) after pre-clearing.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    GSK3β-mediated phosphorylation of USF2 affects its transactivity, DNA binding and target gene expression. (A) HeLa cells were cotransfected with pFR-5Gal4-RE-Luc, an expression vector for constitutively active GSK3β-S9A and WT or mutant pcDNA6-Gal4-USF2 (1-231) or the appropriate empty Gal4 vector. The luciferase activity was calculated as fold induction compared to the Gal4-USF2 (1-231)-WT luciferase activity after subtracting the values from the empty Gal4 expression vector. *, significant differences control vs. GSK3β. (B) Representative Western blot of the transfected constructs. 50 µg of protein from transfected cells were probed with an antibody against Gal4, HA-tag and α-tubulin. (C) Quantitative RT-PCR analyses of FAS, HO-1, PAI-1 and USF2 mRNA levels in GSK3β +/+ and GSK3β −/− cells. *, significant differences WT vs. GSK3β −/− . (D) Western Blot analyses of FAS, HO-1 and PAI-1 expression in GSK3β +/+ and GSK3β −/− cells . 50 µg of protein were subjected to Western analysis with antibodies against FAS, HO-1, PAI-1 or β-catenin, c-Myc or α-tubulin; the latter served as a loading control. (E) ChIP was performed in GSK3β +/+ and GSK3β −/− cells with either USF2 antibody, control IgG or RNA Pol II antibody. The quantitative PCR was performed with primers amplifying the FAS, HO-1, and PAI-1 promoter containing the USF2 binding sites, and with primers amplifying the β-actin promoter binding RNA Pol II as outlined in Materials and Methods . *, significant differences WT vs. GSK3β −/− .

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: GSK3β-mediated phosphorylation of USF2 affects its transactivity, DNA binding and target gene expression. (A) HeLa cells were cotransfected with pFR-5Gal4-RE-Luc, an expression vector for constitutively active GSK3β-S9A and WT or mutant pcDNA6-Gal4-USF2 (1-231) or the appropriate empty Gal4 vector. The luciferase activity was calculated as fold induction compared to the Gal4-USF2 (1-231)-WT luciferase activity after subtracting the values from the empty Gal4 expression vector. *, significant differences control vs. GSK3β. (B) Representative Western blot of the transfected constructs. 50 µg of protein from transfected cells were probed with an antibody against Gal4, HA-tag and α-tubulin. (C) Quantitative RT-PCR analyses of FAS, HO-1, PAI-1 and USF2 mRNA levels in GSK3β +/+ and GSK3β −/− cells. *, significant differences WT vs. GSK3β −/− . (D) Western Blot analyses of FAS, HO-1 and PAI-1 expression in GSK3β +/+ and GSK3β −/− cells . 50 µg of protein were subjected to Western analysis with antibodies against FAS, HO-1, PAI-1 or β-catenin, c-Myc or α-tubulin; the latter served as a loading control. (E) ChIP was performed in GSK3β +/+ and GSK3β −/− cells with either USF2 antibody, control IgG or RNA Pol II antibody. The quantitative PCR was performed with primers amplifying the FAS, HO-1, and PAI-1 promoter containing the USF2 binding sites, and with primers amplifying the β-actin promoter binding RNA Pol II as outlined in Materials and Methods . *, significant differences WT vs. GSK3β −/− .

    Article Snippet: In brief, after cross-linking, cell lysis and sonication, chromatin samples were incubated with anti-USF2 antibody (N-18, Cat.Nr. sc-861, Santa Cruz Biotechnology), anti-RNA polymerase II antibody (N-20, Cat.Nr. sc-899X, Santa Cruz Biotechnology) or IgG preimmune serum (BioScience) in an ultrasonicator bath for 1 h at 4°C; after centrifugation, the precleared samples were mixed with protein G Sepharose beads for 1 h at 4°C.

    Techniques: Binding Assay, Expressing, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Western Blot, Transfection, Construct, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Identification of the transcription starting site by 5′RACE PCR. (A) Agarose gel showed multiple 5′RACE PCR products. (B) Mapping the potential TSS of the DRAM gene . The arrow indicated the nested reverse 5′RACE primer. The GeneRacer PCR, based on RNA ligase-mediated and oligo-capping rapid amplification of cDNA, was carried out according to the manufacturer's instructions. The PCR products were resolved on agarose gel, and potential bands were collected and confirmed by DNA sequencing. * indicates a non-specific amplification of the primer dimmers.

    Journal: PLoS ONE

    Article Title: Serum Starvation Induces DRAM Expression in Liver Cancer Cells via Histone Modifications within Its Promoter Locus

    doi: 10.1371/journal.pone.0050502

    Figure Lengend Snippet: Identification of the transcription starting site by 5′RACE PCR. (A) Agarose gel showed multiple 5′RACE PCR products. (B) Mapping the potential TSS of the DRAM gene . The arrow indicated the nested reverse 5′RACE primer. The GeneRacer PCR, based on RNA ligase-mediated and oligo-capping rapid amplification of cDNA, was carried out according to the manufacturer's instructions. The PCR products were resolved on agarose gel, and potential bands were collected and confirmed by DNA sequencing. * indicates a non-specific amplification of the primer dimmers.

    Article Snippet: The protein-DNA complexes were immunoprecipitated with 4 µg antibodies RNA polymerase II (SC-9001, Santa Cruz Biotechnology), and Brg1 (SC-10768); dimethyl-H3-K9 (ab1220, Abcam), tetra-acetyl-H4 (06-866, Upstate) and diacetyl-H3 (07-593, Upstate); and trimethyl-H3-K4 (9751, Cell Signaling Technology).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, DNA Sequencing

    Gene body methylation of METTL7A in thryoid cancer cell lines (A) The enrichment of RNA pol II and MBD2 in gene body of exogenous linear METTL7A with wild type (WT) and mutant (MT) +4919 CpG site in BCPAP and nthy-ori 3-1 thyroid cell lines. (B, C) Methylation level of the exogenous METTL7A template with wild type and mutant +4919 CpG site in nthy-ori 3-1 and BCPAP cell lines. (D) The transcriptional level from exogenous METTL7A with wild type (WT) and mutant (MT) in nthy-ori 3-1 and BCPAP cell lines.

    Journal: Oncotarget

    Article Title: DNA methylation of METTL7A gene body regulates its transcriptional level in thyroid cancer

    doi: 10.18632/oncotarget.16147

    Figure Lengend Snippet: Gene body methylation of METTL7A in thryoid cancer cell lines (A) The enrichment of RNA pol II and MBD2 in gene body of exogenous linear METTL7A with wild type (WT) and mutant (MT) +4919 CpG site in BCPAP and nthy-ori 3-1 thyroid cell lines. (B, C) Methylation level of the exogenous METTL7A template with wild type and mutant +4919 CpG site in nthy-ori 3-1 and BCPAP cell lines. (D) The transcriptional level from exogenous METTL7A with wild type (WT) and mutant (MT) in nthy-ori 3-1 and BCPAP cell lines.

    Article Snippet: Antibodies against RNA polymerase II (RNA pol II), MBD2 and EZH2 were purchased from Santa Cruz.

    Techniques: Methylation, Mutagenesis

    DNA methylation downstream of SALL4 TSS interferes with RNA polymerase II elongation

    Journal: Oncogene

    Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

    doi: 10.1038/onc.2016.399

    Figure Lengend Snippet: DNA methylation downstream of SALL4 TSS interferes with RNA polymerase II elongation

    Article Snippet: The following antibodies were used in this study: SALL4 antibody (Abcam, Cambridge, MA); HBc antibody (Dako, Carpinteria, CA); RNA polymerase II antibody (Santa cruz biotechnology, Dallas, Texas); BRG1 antibody (Abcam, Cambridge, MA); STAT3 antibody (Santa Cruz biotechnology, Dallas, Texas); OCT4 antibody (Abcam, Cambridge, MA); H3K27me3 antibody (Abcam, Cambridge, MA); Histone 3 antibody (Active Motif, Carlsbad, CA).

    Techniques: DNA Methylation Assay

    Activation of an alternative Mef2c promoter during photoreceptor maturation. Binding of RNA Pol II and acetylation of histones at the retinal Mef2c promoter were measured by ChIP-qPCR using a Pol-II-S2 antibody recognizing the active form of Pol II (

    Journal: The Journal of Biological Chemistry

    Article Title: The Transcription Factor Neural Retina Leucine Zipper (NRL) Controls Photoreceptor-specific Expression of Myocyte Enhancer Factor Mef2c from an Alternative Promoter

    doi: 10.1074/jbc.M111.271072

    Figure Lengend Snippet: Activation of an alternative Mef2c promoter during photoreceptor maturation. Binding of RNA Pol II and acetylation of histones at the retinal Mef2c promoter were measured by ChIP-qPCR using a Pol-II-S2 antibody recognizing the active form of Pol II (

    Article Snippet: The following ChIP grade antibodies were used: anti-Pol II, a polyclonal antibody to total RNA polymerase II (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Pol-II-S2, an antibody against transcriptionally active Pol II, phosphorylated on serine 2 of the C-terminal repeat domain (Abcam Inc., Cambridge, MA); anti-H3K9-Ac, a polyclonal antibody to histone H3 acetylated on lysine 9 (Abcam Inc.); and anti-NRL polyclonal antibody ( ).

    Techniques: Activation Assay, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) RNA Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated DNA was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Repression of Ccr9 transcription in mouse T lymphocyte progenitors by the Notch signaling pathway

    doi: 10.4049/jimmunol.1402443

    Figure Lengend Snippet: Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) RNA Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated DNA was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p

    Article Snippet: The protein-DNA complexes were detected using 5 μg of antibody against either RNA Pol II (N-20)(Santa Cruz, sc-899x), p300 (C-20)(Santa Cruz, sc-585x), HEB (A-20, Santa Cruz, sc-357) or E2A (N-649, Santa Cruz, sc-763x) in one ml of 0.8 M RIPA and incubated overnight at 4°C under rotating conditions.

    Techniques: Chromatin Immunoprecipitation, Isolation, Amplification, Real-time Polymerase Chain Reaction

    H2A.Z is required for the antiproliferative effect of HDACi. a) Cell growth assay b) Trypan blue assay c) qPCR analysis of p21 mRNA expression. d) ChIP analysis of Acetyl-H2A.Z. e) ChIP analysis of RNA pol II. MDA-MB231cells were treated or not with TSA (50 ng/ml) for 24 h/48 h and/or transfected with a smartpool siH2A.Z (72 h) as indicated.

    Journal: PLoS ONE

    Article Title: Activation of p21 by HDAC Inhibitors Requires Acetylation of H2A.Z

    doi: 10.1371/journal.pone.0054102

    Figure Lengend Snippet: H2A.Z is required for the antiproliferative effect of HDACi. a) Cell growth assay b) Trypan blue assay c) qPCR analysis of p21 mRNA expression. d) ChIP analysis of Acetyl-H2A.Z. e) ChIP analysis of RNA pol II. MDA-MB231cells were treated or not with TSA (50 ng/ml) for 24 h/48 h and/or transfected with a smartpool siH2A.Z (72 h) as indicated.

    Article Snippet: Chromatin fragments were immunoprecipitated using antibodies against H2A.Z (ab4174, ABCAM), Acetyl H2A.Z (ab18262, ABCAM), RNA pol II (N20X, Santa Cruz), H3 (ab1791, ABCAM), Acetyl H3K9 (06–942, Millipore), TIP60 , p400 (ab70301, ABCAM), p300 (SC-584, SantaCruz Biotechnology) or an irrelevant HA antibody (H6908, Sigma) as control.

    Techniques: Growth Assay, Real-time Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Multiple Displacement Amplification, Transfection

    p400 but not Tip60 functions in p21 expression in the absence of p53. a) Tip60 and p21 mRNA expression. b) H2A.Z, Acetyl-H2A.Z and RNA pol II binding to the p21 TSS in cell transfected with siTip60 or scramble siRNA. c) ChIP analysis of Tip60 and p400 recruitment to the p21 TSS (fragment #4) in cells treated with TSA. d) p21 mRNA expression in MDA-MB231 transfected with siRNA against p400 and treated or not with TSA. e) H2A.Z, Acetyl-H2A.Z and p300 enrichment at p21 TSS (fragment #4) in MDA-MB231 transfected with si p400.

    Journal: PLoS ONE

    Article Title: Activation of p21 by HDAC Inhibitors Requires Acetylation of H2A.Z

    doi: 10.1371/journal.pone.0054102

    Figure Lengend Snippet: p400 but not Tip60 functions in p21 expression in the absence of p53. a) Tip60 and p21 mRNA expression. b) H2A.Z, Acetyl-H2A.Z and RNA pol II binding to the p21 TSS in cell transfected with siTip60 or scramble siRNA. c) ChIP analysis of Tip60 and p400 recruitment to the p21 TSS (fragment #4) in cells treated with TSA. d) p21 mRNA expression in MDA-MB231 transfected with siRNA against p400 and treated or not with TSA. e) H2A.Z, Acetyl-H2A.Z and p300 enrichment at p21 TSS (fragment #4) in MDA-MB231 transfected with si p400.

    Article Snippet: Chromatin fragments were immunoprecipitated using antibodies against H2A.Z (ab4174, ABCAM), Acetyl H2A.Z (ab18262, ABCAM), RNA pol II (N20X, Santa Cruz), H3 (ab1791, ABCAM), Acetyl H3K9 (06–942, Millipore), TIP60 , p400 (ab70301, ABCAM), p300 (SC-584, SantaCruz Biotechnology) or an irrelevant HA antibody (H6908, Sigma) as control.

    Techniques: Expressing, Binding Assay, Transfection, Chromatin Immunoprecipitation, Multiple Displacement Amplification

    Chromatin immunoprecipitation assay performed on MCF7, and MDA-MB231 breast cancer cell lines to evaluate in vivo Sp1 binding . Proteins were cross-linked to the DNA with formaldehyde and Abs directed against RNA-Pol (Ab control) or Sp1 were added to precipitate any protein-DNA complexes. The control + lane was DNA that had been sonicated and pre-cleared with protein G beads. The control-lanes were processed according to the protocol, but did not have any Ab added to the samples. PCR were performed on isolated DNA using primers encompassing MsrB1 promoter region -169 to +76. A schematic map of the amplified DNA fragment (245 bp) containing Sp1 binding motifs and TSS position is illustrated as well.

    Journal: BMC Molecular Biology

    Article Title: Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter

    doi: 10.1186/1471-2199-8-39

    Figure Lengend Snippet: Chromatin immunoprecipitation assay performed on MCF7, and MDA-MB231 breast cancer cell lines to evaluate in vivo Sp1 binding . Proteins were cross-linked to the DNA with formaldehyde and Abs directed against RNA-Pol (Ab control) or Sp1 were added to precipitate any protein-DNA complexes. The control + lane was DNA that had been sonicated and pre-cleared with protein G beads. The control-lanes were processed according to the protocol, but did not have any Ab added to the samples. PCR were performed on isolated DNA using primers encompassing MsrB1 promoter region -169 to +76. A schematic map of the amplified DNA fragment (245 bp) containing Sp1 binding motifs and TSS position is illustrated as well.

    Article Snippet: No antibodies were added to one aliquot (negative control), and either RNA pol II antibody or Sp1-specific antibody (Santa Cruz, sc-14027 X) was added to the others two aliquots and they incubated overnight at 4°C on a rotating wheel.

    Techniques: Chromatin Immunoprecipitation, Multiple Displacement Amplification, In Vivo, Binding Assay, Sonication, Polymerase Chain Reaction, Isolation, Amplification

    Analysis of eEF1γ depletion. A : Indirect immunofluorescence obtained with the anti-Tom20 rabbit polyclonal and the anti-Vimentin monoclonal antibody in HeLa cells treated with siRNA-Control or with siRNA-eEF1γ. On the right side a higher magnification image of eEF1γ-depleted HeLa cells focusing on the formation of mitochondrial dysfunctional clusters. B : Real time RT-PCR analysis of the eEF1γ (left) and Vimentin (right) mRNAs in HeLa cells (siRNA-Control and siRNA-eEF1γ). The gene expression ratio between eEF1γ and GAPDH and between Vimentin and GAPDH are shown as means ± SD from three independent experiments performed in triplicate. C : The global protein synthesis of HeLa cells, transfected with either siRNA-Control or siRNA-eEF1γ and supplemented with S 35 labelled methionin and cystein, was visualized by autoradiography of the total protein lysates blotted to nitrocellulose membrane. Then the same membrane was incubated with the anti-eEF1γ rabbit polyclonal and the anti-Vimentin mouse monoclonal antibodies. Anti-β-actin monoclonal antibody was used to normalise the amount of protein loaded on the gel. D : Induced carbonylation pattern (oxidation level) of HeLa cells: untreated, treated with either siRNA-Control or siRNA-eEF1γ. The total protein carbonylation pattern was visualised by western blot with the anti-DNP antibody. Depletion of eEF1γ by siRNA was monitored using the anti-eEF1γ rabbit polyclonal antibody. The levels of Tom20 protein were monitored using the anti-Tom20 polyclonal antibody. Anti-alpha-tubulin monoclonal antibody was used to normalise the amount of protein loaded on the gel. E : Representative florescence images of HeLa cells treated with either siRNA-Control or siRNA-eEF1γ (top). The cellular level of superoxide was visualized by the MitoSOX (red) mitochondrial Superoxide Indicator staining. Nuclei were stained with Hoechst (blue). Histogram reporting fold of increase of MitoSOX fluorescence of siRNA-eEF1γ treated cells (lane 2) versus siRNA-Control treated cells (lane 1), shown as means ± SD from three independent experiments (bottom).

    Journal: PLoS ONE

    Article Title: The eEF1? Subunit Contacts RNA Polymerase II and Binds Vimentin Promoter Region

    doi: 10.1371/journal.pone.0014481

    Figure Lengend Snippet: Analysis of eEF1γ depletion. A : Indirect immunofluorescence obtained with the anti-Tom20 rabbit polyclonal and the anti-Vimentin monoclonal antibody in HeLa cells treated with siRNA-Control or with siRNA-eEF1γ. On the right side a higher magnification image of eEF1γ-depleted HeLa cells focusing on the formation of mitochondrial dysfunctional clusters. B : Real time RT-PCR analysis of the eEF1γ (left) and Vimentin (right) mRNAs in HeLa cells (siRNA-Control and siRNA-eEF1γ). The gene expression ratio between eEF1γ and GAPDH and between Vimentin and GAPDH are shown as means ± SD from three independent experiments performed in triplicate. C : The global protein synthesis of HeLa cells, transfected with either siRNA-Control or siRNA-eEF1γ and supplemented with S 35 labelled methionin and cystein, was visualized by autoradiography of the total protein lysates blotted to nitrocellulose membrane. Then the same membrane was incubated with the anti-eEF1γ rabbit polyclonal and the anti-Vimentin mouse monoclonal antibodies. Anti-β-actin monoclonal antibody was used to normalise the amount of protein loaded on the gel. D : Induced carbonylation pattern (oxidation level) of HeLa cells: untreated, treated with either siRNA-Control or siRNA-eEF1γ. The total protein carbonylation pattern was visualised by western blot with the anti-DNP antibody. Depletion of eEF1γ by siRNA was monitored using the anti-eEF1γ rabbit polyclonal antibody. The levels of Tom20 protein were monitored using the anti-Tom20 polyclonal antibody. Anti-alpha-tubulin monoclonal antibody was used to normalise the amount of protein loaded on the gel. E : Representative florescence images of HeLa cells treated with either siRNA-Control or siRNA-eEF1γ (top). The cellular level of superoxide was visualized by the MitoSOX (red) mitochondrial Superoxide Indicator staining. Nuclei were stained with Hoechst (blue). Histogram reporting fold of increase of MitoSOX fluorescence of siRNA-eEF1γ treated cells (lane 2) versus siRNA-Control treated cells (lane 1), shown as means ± SD from three independent experiments (bottom).

    Article Snippet: Immunoprecipitation assays were performed following standard procedure, using the following antibodies: agarose-covalently attached anti-flag monoclonal M2 (Sigma), anti-myc monoclonal antibody (9E10 clone, hybridoma-conditioned medium) and anti-pol II rabbit polyclonal antibody (Santa Cruz).

    Techniques: Immunofluorescence, Quantitative RT-PCR, Expressing, Transfection, Autoradiography, Incubation, Western Blot, Staining, Fluorescence

    eEF1γ interacts with pol II and binds Vimentin gene promoter region. A : Total lysates from HeLa cells transfected with either an empty control vector or myc-eEF1γ were immunoprecipitated with the anti-pol II polyclonal antibody and analysed by western blot using the anti-myc monoclonal antibody. B : Whole extracts from HeLa cells immunoprecipitated with the anti-pol II polyclonal antibody. Co-immunoprecipitation was analysed by western blot using the anti-eEF1γ polyclonal antibody. The eEF1γ signal is specifically detected in the pol II I.P. sample (below the heavy chain Ig band). The I.P. control was performed with normal rabbit serum. Total cell lysates and immunopreciptated samples were immunoblotted with the anti-pol II polyclonal antibody. C : Western blot analysis of the cytoplasmic and nuclear fractions derived from HeLa cells. The blot was incubated with the anti-eEF1γ polyclonal antibody to determine the subcellular localization of eEF1γ. To verify fractionation quality, the same extracts were incubated with the anti-Hax1, anti-Sp1 and anti-alpha-tubulin antibodies. D : eEF1γ stays on Vimentin promoter at the endogenous chromosomal site. Chromatin immuno-precipitation was performed in HeLa cells using the anti-eEF1γ rabbit polyclonal antibody/protein G-agarose beads or only protein G-agarose beads as a control (no-Ab). Immuno-precipitates from each sample were analysed by PCR performed using primers specific for the human Vimentin promoter. The DNA-pol β and thymidine kinase human promoters were also amplified. A sample representing linear amplification of the total input chromatin (input) was included in the PCR as a control.

    Journal: PLoS ONE

    Article Title: The eEF1? Subunit Contacts RNA Polymerase II and Binds Vimentin Promoter Region

    doi: 10.1371/journal.pone.0014481

    Figure Lengend Snippet: eEF1γ interacts with pol II and binds Vimentin gene promoter region. A : Total lysates from HeLa cells transfected with either an empty control vector or myc-eEF1γ were immunoprecipitated with the anti-pol II polyclonal antibody and analysed by western blot using the anti-myc monoclonal antibody. B : Whole extracts from HeLa cells immunoprecipitated with the anti-pol II polyclonal antibody. Co-immunoprecipitation was analysed by western blot using the anti-eEF1γ polyclonal antibody. The eEF1γ signal is specifically detected in the pol II I.P. sample (below the heavy chain Ig band). The I.P. control was performed with normal rabbit serum. Total cell lysates and immunopreciptated samples were immunoblotted with the anti-pol II polyclonal antibody. C : Western blot analysis of the cytoplasmic and nuclear fractions derived from HeLa cells. The blot was incubated with the anti-eEF1γ polyclonal antibody to determine the subcellular localization of eEF1γ. To verify fractionation quality, the same extracts were incubated with the anti-Hax1, anti-Sp1 and anti-alpha-tubulin antibodies. D : eEF1γ stays on Vimentin promoter at the endogenous chromosomal site. Chromatin immuno-precipitation was performed in HeLa cells using the anti-eEF1γ rabbit polyclonal antibody/protein G-agarose beads or only protein G-agarose beads as a control (no-Ab). Immuno-precipitates from each sample were analysed by PCR performed using primers specific for the human Vimentin promoter. The DNA-pol β and thymidine kinase human promoters were also amplified. A sample representing linear amplification of the total input chromatin (input) was included in the PCR as a control.

    Article Snippet: Immunoprecipitation assays were performed following standard procedure, using the following antibodies: agarose-covalently attached anti-flag monoclonal M2 (Sigma), anti-myc monoclonal antibody (9E10 clone, hybridoma-conditioned medium) and anti-pol II rabbit polyclonal antibody (Santa Cruz).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Derivative Assay, Incubation, Fractionation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

    eEF1γ partially co-localizes with mitochondria and its depletion influences Vimentin protein localisation. A : Indirect immunofluorescence of Vimentin, obtained with the monoclonal antibody anti-Vimentin, in HeLa cells: untreated, treated with siRNA-Control and eEF1γ-depleted by specific siRNA. B : Western blot analysis of HeLa cells (from panel A): untreated and treated with either siRNA-Control or with eEF1γ specific siRNA. C : Western blot analysis of HeLa cell total lysate and the enriched mitochondrial fraction. The quality of mitochondrial enriched fraction was monitored using the anti-Hax1 monoclonal antibody and the anti-Tom20 rabbit polyclonal antibody. D : Dual-label indirect immunofluorescence performed in HeLa cells with the anti-Tom20 rabbit polyclonal antibody and the anti-eEF1γ monoclonal antibody to visualise the immunolocalisation of endogenous eEF1γ (green) and Tom20 (red). Extensive co-localization (yellow) between eEF1γ and Tom20 is visualised by the merged-colour image. On the right side, the same immunofluorescences are presented at a higher magnification (100×).

    Journal: PLoS ONE

    Article Title: The eEF1? Subunit Contacts RNA Polymerase II and Binds Vimentin Promoter Region

    doi: 10.1371/journal.pone.0014481

    Figure Lengend Snippet: eEF1γ partially co-localizes with mitochondria and its depletion influences Vimentin protein localisation. A : Indirect immunofluorescence of Vimentin, obtained with the monoclonal antibody anti-Vimentin, in HeLa cells: untreated, treated with siRNA-Control and eEF1γ-depleted by specific siRNA. B : Western blot analysis of HeLa cells (from panel A): untreated and treated with either siRNA-Control or with eEF1γ specific siRNA. C : Western blot analysis of HeLa cell total lysate and the enriched mitochondrial fraction. The quality of mitochondrial enriched fraction was monitored using the anti-Hax1 monoclonal antibody and the anti-Tom20 rabbit polyclonal antibody. D : Dual-label indirect immunofluorescence performed in HeLa cells with the anti-Tom20 rabbit polyclonal antibody and the anti-eEF1γ monoclonal antibody to visualise the immunolocalisation of endogenous eEF1γ (green) and Tom20 (red). Extensive co-localization (yellow) between eEF1γ and Tom20 is visualised by the merged-colour image. On the right side, the same immunofluorescences are presented at a higher magnification (100×).

    Article Snippet: Immunoprecipitation assays were performed following standard procedure, using the following antibodies: agarose-covalently attached anti-flag monoclonal M2 (Sigma), anti-myc monoclonal antibody (9E10 clone, hybridoma-conditioned medium) and anti-pol II rabbit polyclonal antibody (Santa Cruz).

    Techniques: Immunofluorescence, Western Blot

    Knockdown of EPHB3 rescues growth of TCF7L1-Null cells. ( A ) Immunohistochemistry for EPHB3 shows elevated expression in TCF7L1-Null xenograft tumors (bottom) compared to control tumors from the same mice (top), reflecting the significant mRNA upregulation observed by RNA-sequencing and qPCR. ( B ) EPHB3 knockdown largely rescued colony formation of TCF7L1-Null cells. Colony number and average colony size was significantly higher than that of TCF7L1-Null cells, and almost returned to the levels of control cells (*P

    Journal: Scientific Reports

    Article Title: TCF7L1 Modulates Colorectal Cancer Growth by Inhibiting Expression of the Tumor-Suppressor Gene EPHB3

    doi: 10.1038/srep28299

    Figure Lengend Snippet: Knockdown of EPHB3 rescues growth of TCF7L1-Null cells. ( A ) Immunohistochemistry for EPHB3 shows elevated expression in TCF7L1-Null xenograft tumors (bottom) compared to control tumors from the same mice (top), reflecting the significant mRNA upregulation observed by RNA-sequencing and qPCR. ( B ) EPHB3 knockdown largely rescued colony formation of TCF7L1-Null cells. Colony number and average colony size was significantly higher than that of TCF7L1-Null cells, and almost returned to the levels of control cells (*P

    Article Snippet: Gels were transferred onto nitrocellulose membranes and incubated overnight at 4C with one or more of the following antibodies: mouse anti-β-catenin (Sigma 15B8, 1:1,000), rabbit anti-TCF7L1 (Cell Signaling D15G11, 1:200), rabbit anti-LEF1 (Cell Signaling C12A5, 1:200), rabbit anti-TCF7 (Cell Signaling C63D9, 1:200), rabbit anti-TCF7L2 (Cell Signaling C48H11, 1:200), rabbit anti-EPHB3 (abcam EPR8280, 1:200), mouse anti-tubulin (Sigma T9026, 1:500), rabbit anti-RNA Pol II (Santa Cruz N-20, 1:200), mouse anti-GAPDH (Santa Cruz 6C5, 1:500), rabbit anti-Na + /K + -ATPase α (Santa Cruz H-300, 1:200).

    Techniques: Immunohistochemistry, Expressing, Mouse Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    Alteration of Cx43 promoter associated proteins by vorinostat . (A) Illustration of the Cx43 promoter region up to -0.5 kb from the Cx43 gene ( Gja1 ) transcription start site of Cx43 with TATA and GC boxes indicated. Two primer sets were designed from -511 to -206 and -225 to +65 bp of the Cx43 promoter region. (B,C) Chromatin immunoprecipitation assays illustrate the reduced binding of RNA polymerase II (RNA Pol II) and specific protein 1 (Sp1) to the promoter region of Cx43 after overnight exposure to 2 μM VOR. (D,E) A chromatin immunoprecipitation assay was performed with rabbit IgG and rabbit anti-HDAC1 or anti-HDAC2 antibodies to assess the binding of HDAC1 (D) and HDAC2 (E) to the promoter region of the Cx43 gene ( Gja1 ). Overnight treatment with 2 μM VOR enhanced the association of HDAC1 and HDAC2 to the Gja1 promoter.

    Journal: Frontiers in Pharmacology

    Article Title: Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication

    doi: 10.3389/fphar.2013.00044

    Figure Lengend Snippet: Alteration of Cx43 promoter associated proteins by vorinostat . (A) Illustration of the Cx43 promoter region up to -0.5 kb from the Cx43 gene ( Gja1 ) transcription start site of Cx43 with TATA and GC boxes indicated. Two primer sets were designed from -511 to -206 and -225 to +65 bp of the Cx43 promoter region. (B,C) Chromatin immunoprecipitation assays illustrate the reduced binding of RNA polymerase II (RNA Pol II) and specific protein 1 (Sp1) to the promoter region of Cx43 after overnight exposure to 2 μM VOR. (D,E) A chromatin immunoprecipitation assay was performed with rabbit IgG and rabbit anti-HDAC1 or anti-HDAC2 antibodies to assess the binding of HDAC1 (D) and HDAC2 (E) to the promoter region of the Cx43 gene ( Gja1 ). Overnight treatment with 2 μM VOR enhanced the association of HDAC1 and HDAC2 to the Gja1 promoter.

    Article Snippet: The pre-cleared cell lysis was immunoprecipitated with 10 μg of rabbit anti-HDAC1 antibody (Enzo Life Sciences), rabbit anti-HDAC2 antibody (Enzo Life Sciences), rabbit anti-Sp1 antibody (Santa Cruz), rabbit anti-RNA Pol II antibody (Santa Cruz), or normal rabbit IgG (Sigma) overnight (4°C).

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    DDX5 localizes to promoters of DNA replication genes and functions in the loading of RNA Polymerase II onto these promoters. (A) Western blot analysis of E2F1 and DDX5 in DDX5 and IgG immunoprecipitation samples from nuclear extracts prepared from HCT116 cultures (top) and analysis of the interaction of in vitro transcribed and translated radiolabeled DDX5 with GST-E2F1 purified from bacteria and immobilized on glutathione beads (bottom). (B) Q-PCR analysis of DDX5 ChIP samples at the indicated promoters. Cells were serum starved, then re-stimulated with serum for 2 hrs and then harvested for ChIP. Note that the CD4 promoter is used as negative control. Error bars show the standard deviations for duplicate experiments. The green bars correspond to results for the DDX5 ChIPs whereas the black bars correspond to results for the IgG control ChIPs. All primer pairs used for Q-PCR amplify within 200bp of the transcription start sites of the indicated genes. Q-PCR analysis of E2F1 ChIP (C), Acetylated Histone H3 ChIP (D), RNA Polymerase II ChIP (E), and TFIIB ChIP (F) at the indicated promoters in asynchronous cells transfected with either DDX5si2008 (blue bars) or EBNA1si1666 (red bars) siRNAs. Error bars show the standard deviations for duplicate experiments. Neither the GAPDH nor CD4 transcripts are down-regulated by DDX5 knockdown and therefore their promoters are used as negative controls in for the ChIP experiments presented in (D), (E), and (F).

    Journal: Cancer discovery

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells

    doi: 10.1158/2159-8290.CD-12-0116

    Figure Lengend Snippet: DDX5 localizes to promoters of DNA replication genes and functions in the loading of RNA Polymerase II onto these promoters. (A) Western blot analysis of E2F1 and DDX5 in DDX5 and IgG immunoprecipitation samples from nuclear extracts prepared from HCT116 cultures (top) and analysis of the interaction of in vitro transcribed and translated radiolabeled DDX5 with GST-E2F1 purified from bacteria and immobilized on glutathione beads (bottom). (B) Q-PCR analysis of DDX5 ChIP samples at the indicated promoters. Cells were serum starved, then re-stimulated with serum for 2 hrs and then harvested for ChIP. Note that the CD4 promoter is used as negative control. Error bars show the standard deviations for duplicate experiments. The green bars correspond to results for the DDX5 ChIPs whereas the black bars correspond to results for the IgG control ChIPs. All primer pairs used for Q-PCR amplify within 200bp of the transcription start sites of the indicated genes. Q-PCR analysis of E2F1 ChIP (C), Acetylated Histone H3 ChIP (D), RNA Polymerase II ChIP (E), and TFIIB ChIP (F) at the indicated promoters in asynchronous cells transfected with either DDX5si2008 (blue bars) or EBNA1si1666 (red bars) siRNAs. Error bars show the standard deviations for duplicate experiments. Neither the GAPDH nor CD4 transcripts are down-regulated by DDX5 knockdown and therefore their promoters are used as negative controls in for the ChIP experiments presented in (D), (E), and (F).

    Article Snippet: For ChIP experiments rabbit anti-DDX5 from Bethyl Laboratories cat. # A300-523A, Mouse anti-E2F1 from Sigma cat. # E8901, Rabbit anti-acetyl-Histone H3 from Millipore cat. # 06-599, Rabbit anti-RNA Polymerase II from Santa Cruz cat. # sc-899, and Rabbit anti-TFIIB from Santa Cruz cat. # sc-225X were used.

    Techniques: Western Blot, Immunoprecipitation, In Vitro, Purification, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Transfection