ctcf antibody  (Millipore)


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    Name:
    Anti CTCF antibody
    Description:

    Catalog Number:
    hpa004122
    Price:
    None
    Applications:
    All Prestige Antibodies Powered by Atlas Antibodies are developed and validated by the Human Protein Atlas (HPA) project (www.proteinatlas.org)and as a result, are supported by the most extensive characterization in the industry. The Human Protein Atlas project can be subdivided into three efforts: Human Tissue Atlas, Cancer Atlas, and Human Cell Atlas. The antibodies that have been generated in support of the Tissue and Cancer Atlas projects have been tested by immunohistochemistry against hundreds of normal and disease tissues and through the recent efforts of the Human Cell Atlas project, many have been characterized by immunofluorescence to map the human proteome not only at the tissue level but now at the subcellular level. These images and the collection of this vast data set can be viewed on the Human Protein Atlas (HPA) site by clicking on the Image Gallery link. To view these protocols and other useful information about Prestige Antibodies and the HPA, visit sigma.com/prestige.
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    Millipore ctcf antibody
    Anti CTCF antibody

    https://www.bioz.com/result/ctcf antibody/product/Millipore
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    ctcf antibody - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "CTCF binding site sequence differences are associated with unique regulatory and functional trends during embryonic stem cell differentiation"

    Article Title: CTCF binding site sequence differences are associated with unique regulatory and functional trends during embryonic stem cell differentiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt910

    CTCF binding during ES cell differentiation. Representative CTCF ChIP-Seq peaks where binding is lost ( A ), maintained ( B ) and gained ( C ) during differentiation are shown. Y-axis shows relative enrichment above IgG. The relative proportion of LowOc, MedOc and HighOc sites among sites that are lost, gained or maintained during ES cell differentiation are also shown ( D ). The proportion of sites from each class was compared between the different groups via Fisher's exact test. LowOc sites are significantly enriched among sites where CTCF binding is lost ( P = E-81) and gained ( P = E-18) as compared with those that maintain binding.
    Figure Legend Snippet: CTCF binding during ES cell differentiation. Representative CTCF ChIP-Seq peaks where binding is lost ( A ), maintained ( B ) and gained ( C ) during differentiation are shown. Y-axis shows relative enrichment above IgG. The relative proportion of LowOc, MedOc and HighOc sites among sites that are lost, gained or maintained during ES cell differentiation are also shown ( D ). The proportion of sites from each class was compared between the different groups via Fisher's exact test. LowOc sites are significantly enriched among sites where CTCF binding is lost ( P = E-81) and gained ( P = E-18) as compared with those that maintain binding.

    Techniques Used: Binding Assay, Cell Differentiation, Chromatin Immunoprecipitation

    2) Product Images from "Mapping and Functional Characterisation of a CTCF-Dependent Insulator Element at the 3? Border of the Murine Scl Transcriptional Domain"

    Article Title: Mapping and Functional Characterisation of a CTCF-Dependent Insulator Element at the 3? Border of the Murine Scl Transcriptional Domain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031484

    In vivo binding of CTCF to 57FPR sequence in haematopoietic progenitor cells. ( A ) DMS footprinting of the 350 base pair core region of the +45 element in 3 cell types, BW5147, 416B and mouse ES cells, shows a DNA protein footprint that extends to 57 base pairs in length. Open circles represent relative protection from DMS-induced methylation compared with naked genomic DNA control, while the closed circle represents enhancement of DMS-induced methylation. Multiple lanes correspond to different biological replicates. The sequence of the 57 bp footprinted region (57FPR) is shown with cross-species sequence comparison. The CTCF-consensus binding site from Xie et al is also represented. ( B ) EMSA demonstrating a direct interaction between the 57FPR and CTCF protein (lane 2). The interaction is lost by competition with wild type 57FPR (lane 3), but not by CTCF-mutated 57FPR (lane 4). The arrow indicates the super shifted complex by co-incubation with specific anti-CTCF antibody (lane 5). The wild type and mutated sequence used for the EMSA are also shown. ( C ). ChIP-chip profiles mapping CTCF binding across 150 kb of the extended Scl locus in 2 cell types, 416B (mouse haematopoietic progenitor cells) and mouse embryonic stem cells (mES). The location of the 4 genes at the locus are represented at the top of the figure (Sil, Scl, Map17 and Cyp4x) with direction of transcription indicated by the arrows and translated and untranslated exons represented by wide and narrow bars respectively. The location of certain known key regulatory elements is indicated by the red bars (Sil promoter, Scl promoter, Scl+19, Scl+23, Map17 promoter, Scl+40 and Cyp promoter), and the location of the putative +45 element is indicated with a dashed red line. Representative experiment is showing fold enrichment over non-enriched input plotted (log 2 ) against genomic position. In the cell types studied, the +45 element is the most highly enriched site for CTCF across the extended Scl locus.
    Figure Legend Snippet: In vivo binding of CTCF to 57FPR sequence in haematopoietic progenitor cells. ( A ) DMS footprinting of the 350 base pair core region of the +45 element in 3 cell types, BW5147, 416B and mouse ES cells, shows a DNA protein footprint that extends to 57 base pairs in length. Open circles represent relative protection from DMS-induced methylation compared with naked genomic DNA control, while the closed circle represents enhancement of DMS-induced methylation. Multiple lanes correspond to different biological replicates. The sequence of the 57 bp footprinted region (57FPR) is shown with cross-species sequence comparison. The CTCF-consensus binding site from Xie et al is also represented. ( B ) EMSA demonstrating a direct interaction between the 57FPR and CTCF protein (lane 2). The interaction is lost by competition with wild type 57FPR (lane 3), but not by CTCF-mutated 57FPR (lane 4). The arrow indicates the super shifted complex by co-incubation with specific anti-CTCF antibody (lane 5). The wild type and mutated sequence used for the EMSA are also shown. ( C ). ChIP-chip profiles mapping CTCF binding across 150 kb of the extended Scl locus in 2 cell types, 416B (mouse haematopoietic progenitor cells) and mouse embryonic stem cells (mES). The location of the 4 genes at the locus are represented at the top of the figure (Sil, Scl, Map17 and Cyp4x) with direction of transcription indicated by the arrows and translated and untranslated exons represented by wide and narrow bars respectively. The location of certain known key regulatory elements is indicated by the red bars (Sil promoter, Scl promoter, Scl+19, Scl+23, Map17 promoter, Scl+40 and Cyp promoter), and the location of the putative +45 element is indicated with a dashed red line. Representative experiment is showing fold enrichment over non-enriched input plotted (log 2 ) against genomic position. In the cell types studied, the +45 element is the most highly enriched site for CTCF across the extended Scl locus.

    Techniques Used: In Vivo, Binding Assay, Sequencing, Footprinting, Methylation, Incubation, Chromatin Immunoprecipitation

    3) Product Images from "The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation"

    Article Title: The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0618-0

    Knockdown of Firre or Ctcf decreases association of Firre and Dxz4 to the nucleolus. (A) Association frequency (in % of nuclei) between Firre or Dxz4 and the nucleolus as determined by DNA-FISH and immunostaining is significantly reduced after either Firre or Ctcf knockdown in Patski cells. At least 100 nuclei were scored in each experiment. P values were determined by two-tail unpaired student t-test. Error bars represent s.e.m. (B) Occupancy by CTCF and RAD21 at Firre and Dxz4 before and after knockdowns of Firre or Ctcf . ChIP enrichment is shown as the ratio between ChIP and input fractions as measured by qRT-PCR. Error bars represent s.e.m.
    Figure Legend Snippet: Knockdown of Firre or Ctcf decreases association of Firre and Dxz4 to the nucleolus. (A) Association frequency (in % of nuclei) between Firre or Dxz4 and the nucleolus as determined by DNA-FISH and immunostaining is significantly reduced after either Firre or Ctcf knockdown in Patski cells. At least 100 nuclei were scored in each experiment. P values were determined by two-tail unpaired student t-test. Error bars represent s.e.m. (B) Occupancy by CTCF and RAD21 at Firre and Dxz4 before and after knockdowns of Firre or Ctcf . ChIP enrichment is shown as the ratio between ChIP and input fractions as measured by qRT-PCR. Error bars represent s.e.m.

    Techniques Used: Fluorescence In Situ Hybridization, Immunostaining, Chromatin Immunoprecipitation, Quantitative RT-PCR

    The Firre/FIRRE loci bind CTCF and RAD21 in mouse and human females. (A) Schematic of the mouse X chromosome showing the location of Firre , and Xist . Blow-up shows the lncRNA Firre (transcription direction marked by an arrow) with the location of segmental duplications of tandem repeats (same color indicates paired duplicated regions). Cen, centromere; Tel, telomere. (B) CTCF and RAD21 are bound to Firre in female liver (FL) but not in male liver (ML). ChIP-chip data are shown as log 2 ChIP/input. Genomic coordinates are shown at top. (C) CTCF, cohesin (SMC3 and RAD21), and YY1 are bound to FIRRE in female (red) but not in male human B-lymphocytes (blue). Peak center tracks (darker color indicates peak strength) from the human ENCODE project [ 45 ]. Genomic coordinates are shown at top.
    Figure Legend Snippet: The Firre/FIRRE loci bind CTCF and RAD21 in mouse and human females. (A) Schematic of the mouse X chromosome showing the location of Firre , and Xist . Blow-up shows the lncRNA Firre (transcription direction marked by an arrow) with the location of segmental duplications of tandem repeats (same color indicates paired duplicated regions). Cen, centromere; Tel, telomere. (B) CTCF and RAD21 are bound to Firre in female liver (FL) but not in male liver (ML). ChIP-chip data are shown as log 2 ChIP/input. Genomic coordinates are shown at top. (C) CTCF, cohesin (SMC3 and RAD21), and YY1 are bound to FIRRE in female (red) but not in male human B-lymphocytes (blue). Peak center tracks (darker color indicates peak strength) from the human ENCODE project [ 45 ]. Genomic coordinates are shown at top.

    Techniques Used: Chromatin Immunoprecipitation

    4) Product Images from "PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type"

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type

    Journal: Journal of Virology

    doi: 10.1128/JVI.00755-18

    PARP1 colocalizes with CTCF across the Epstein-Barr virus genome. (A) ChIP-seq for PARP1 and CTCF across the EBV genome in LCLs demonstrating widespread colocalization. Peaks are expressed as counts per million reads. Corresponding genes in the linearized EBV genome are shown below. (B) Zoomed images of PARP1 and CTCF ChIP-seq at Cp, Qp, Zp, and LMP1/2 loci. Peaks are shown as counts per million reads. Scale in the y axes are independent among the loci shown. (C) Western blot showing PARP1 and CTCF interaction in LCLs. Cell lysates were subjected to immunoprecipitation with antibodies for IgG, PARP1, and CTCF. Immune complexes were resolved by gel electrophoresis and immunoblotted for PARP1. (D) ChIP-qPCR for poly(ADP-ribose) moieties at Cp, Qp, Zp, and LMP1 in representative type I (white bars; Mutu, Kem I) and type III (black bars; Kem III, LCL) latent cell lines. qPCR data are presented as fold above the level for IgG. Results are representative of three independent experiments and show means ± standard deviations.
    Figure Legend Snippet: PARP1 colocalizes with CTCF across the Epstein-Barr virus genome. (A) ChIP-seq for PARP1 and CTCF across the EBV genome in LCLs demonstrating widespread colocalization. Peaks are expressed as counts per million reads. Corresponding genes in the linearized EBV genome are shown below. (B) Zoomed images of PARP1 and CTCF ChIP-seq at Cp, Qp, Zp, and LMP1/2 loci. Peaks are shown as counts per million reads. Scale in the y axes are independent among the loci shown. (C) Western blot showing PARP1 and CTCF interaction in LCLs. Cell lysates were subjected to immunoprecipitation with antibodies for IgG, PARP1, and CTCF. Immune complexes were resolved by gel electrophoresis and immunoblotted for PARP1. (D) ChIP-qPCR for poly(ADP-ribose) moieties at Cp, Qp, Zp, and LMP1 in representative type I (white bars; Mutu, Kem I) and type III (black bars; Kem III, LCL) latent cell lines. qPCR data are presented as fold above the level for IgG. Results are representative of three independent experiments and show means ± standard deviations.

    Techniques Used: Chromatin Immunoprecipitation, Western Blot, Immunoprecipitation, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction

    PARP inhibition alters CTCF binding across the Epstein-Barr virus genome. (A) ChIP-seq for CTCF across the EBV genome in untreated or olaparib-treated (PARPi) LCLs and respective input DNA. Peaks are expressed as counts per million reads. Corresponding genes in the linearized EBV genome are shown below. (B) Zoomed image of CTCF ChIP-seq at the latent Cp locus in LCLs, demonstrating the loss of enrichment after olaparib treatment. (C) Independent ChIP-qPCR validation of CTCF enrichment at Cp in untreated or olaparib-treated LCLs. qPCR data are presented as fold above the level for IgG. Results are representative of three independent experiments and show means ± standard deviations. (D) ChIP-qPCR for PARP1 in untreated or olaparib-treated LCLs. qPCR data are presented as fold above the level for IgG. Results are representative of three independent experiments and show means ± standard deviations (ns, not significant).
    Figure Legend Snippet: PARP inhibition alters CTCF binding across the Epstein-Barr virus genome. (A) ChIP-seq for CTCF across the EBV genome in untreated or olaparib-treated (PARPi) LCLs and respective input DNA. Peaks are expressed as counts per million reads. Corresponding genes in the linearized EBV genome are shown below. (B) Zoomed image of CTCF ChIP-seq at the latent Cp locus in LCLs, demonstrating the loss of enrichment after olaparib treatment. (C) Independent ChIP-qPCR validation of CTCF enrichment at Cp in untreated or olaparib-treated LCLs. qPCR data are presented as fold above the level for IgG. Results are representative of three independent experiments and show means ± standard deviations. (D) ChIP-qPCR for PARP1 in untreated or olaparib-treated LCLs. qPCR data are presented as fold above the level for IgG. Results are representative of three independent experiments and show means ± standard deviations (ns, not significant).

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

    In type III latency, CTCF is PARylated by PARP1 at Cp to maintain the open chromatin landscape and transcription. (Top) During type III latency, PARP1 binds along with PARylated CTCF at the active Cp promoter. PARP1 and CTCF cooperate at Cp to promote open chromatin structure and active transcription by maintaining decondensed nucleosomes and H3K4me3 and preventing DNA methylation. (Bottom) PARP inhibition results in the loss of CTCF at Cp and is associated with the formation of a repressive chromatin environment at the Cp promoter. After PARP inhibition, the Cp promoter becomes enriched for DNA methylation, the repressive H3K27me3 mark, and the nucleosomes are compacted, resulting in decreased type III latency transcription.
    Figure Legend Snippet: In type III latency, CTCF is PARylated by PARP1 at Cp to maintain the open chromatin landscape and transcription. (Top) During type III latency, PARP1 binds along with PARylated CTCF at the active Cp promoter. PARP1 and CTCF cooperate at Cp to promote open chromatin structure and active transcription by maintaining decondensed nucleosomes and H3K4me3 and preventing DNA methylation. (Bottom) PARP inhibition results in the loss of CTCF at Cp and is associated with the formation of a repressive chromatin environment at the Cp promoter. After PARP inhibition, the Cp promoter becomes enriched for DNA methylation, the repressive H3K27me3 mark, and the nucleosomes are compacted, resulting in decreased type III latency transcription.

    Techniques Used: DNA Methylation Assay, Inhibition

    5) Product Images from "A role for CTCF and cohesin in subtelomere chromatin organization, TERRA transcription, and telomere end protection"

    Article Title: A role for CTCF and cohesin in subtelomere chromatin organization, TERRA transcription, and telomere end protection

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2012.266

    Enrichment profiles for ChIP-Seq analysis of CTCF, cohesin, and RNAPII binding to human subtelomeres. Fragment density profiles were generated for samples and a matched IgG control as described in Materials and methods. The fold enrichment of sample over
    Figure Legend Snippet: Enrichment profiles for ChIP-Seq analysis of CTCF, cohesin, and RNAPII binding to human subtelomeres. Fragment density profiles were generated for samples and a matched IgG control as described in Materials and methods. The fold enrichment of sample over

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Generated

    6) Product Images from "EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation"

    Article Title: EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation

    Journal: Genome Biology

    doi: 10.1186/s13059-019-1853-6

    EpiMethylTag is a reproducible method for testing whether DNAme can coexist with TF binding (CTCF) or chromatin accessibility genome-wide. a Pearson correlation of read counts comparing M-ATAC with unconverted samples (NC) and regular ATAC-seq (top), and CTCF M-ChIP with unconverted samples, a sample from the Schubeler lab generated using ChIP-BisSeq [ 1 ] (GSE39739) and regular CTCF ChIP-seq (bottom). b Representative IGV screenshots of EpiMethylTag, at the Klf4 locus (left panel), the Pisd-ps1 locus (middle panel), and the Slc5a8 locus (right panel). ATAC and M-ATAC in green, CTCF in purple and DNA methylation from merged M-ATAC, merged CTCF M-ChIP and WGBS (methylation from 0% in blue to 100% in red). A zoom-in of methylation at the highlighted region is shown at the bottom of each example. The Klf4 locus illustrates a region that has low methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Pisd-ps1 locus illustrates a region that has high methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Slc5a8 locus illustrates a region that has low methylation as detected by M-ATAC and high methylation as detected by WGBS. c Density plots of methylation from EpiMethyltag compared with WGBS. Only CpGs inside peaks and with at least five reads were considered. Top: average methylation of CpGs per M-ATAC peak in M-ATAC versus WGBS (Pearson correlation = 0.69, p value
    Figure Legend Snippet: EpiMethylTag is a reproducible method for testing whether DNAme can coexist with TF binding (CTCF) or chromatin accessibility genome-wide. a Pearson correlation of read counts comparing M-ATAC with unconverted samples (NC) and regular ATAC-seq (top), and CTCF M-ChIP with unconverted samples, a sample from the Schubeler lab generated using ChIP-BisSeq [ 1 ] (GSE39739) and regular CTCF ChIP-seq (bottom). b Representative IGV screenshots of EpiMethylTag, at the Klf4 locus (left panel), the Pisd-ps1 locus (middle panel), and the Slc5a8 locus (right panel). ATAC and M-ATAC in green, CTCF in purple and DNA methylation from merged M-ATAC, merged CTCF M-ChIP and WGBS (methylation from 0% in blue to 100% in red). A zoom-in of methylation at the highlighted region is shown at the bottom of each example. The Klf4 locus illustrates a region that has low methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Pisd-ps1 locus illustrates a region that has high methylation as detected by M-ATAC, CTCF M-ChIP, and WGBS. The Slc5a8 locus illustrates a region that has low methylation as detected by M-ATAC and high methylation as detected by WGBS. c Density plots of methylation from EpiMethyltag compared with WGBS. Only CpGs inside peaks and with at least five reads were considered. Top: average methylation of CpGs per M-ATAC peak in M-ATAC versus WGBS (Pearson correlation = 0.69, p value

    Techniques Used: Binding Assay, Genome Wide, Chromatin Immunoprecipitation, Generated, DNA Methylation Assay, Methylation

    M-ChIP enables analysis of DNA methylation binding by CTCF and KLF4. a Top: Schematic illustration representing an ATAC-seq peak with a CTCF motif and CTCF occupancy dependent on C2 and C12 methylation. Bottom: average profiles of M-ATAC (left) and CTCF M-ChIP (right) intensity at CpGs in a CTCF motif within M-ATAC peaks for the four groups of CpGs (group #1: 288 CpGs, group #2: 17133 CpGs, group #3 CpGs: 758, group #4: 25 CpGs). b top: CTCF motif from JASPAR database (MA0139.1). The 2 key CpG positions (C2 and C12) are indicated. Bottom: violin plots of methylation percentage from CTCF M-ChIP and WGBS, at C2 and C12 positions in the CTCF motif (MA0139.1). *** P = 1.02e−12 for C2 CTCF M-ChIP versus C12 CTCF M-ChIP (Wilcoxon test), ** P = 0.008 for C2 WGBS versus C12 WGBS (Wilcoxon test), *** P = 9e−12 for C2 CTCF M-ChIP versus C2 WGBS (Wilcoxon test, paired), *** P = 0.00075 for C12 CTCF M-ChIP versus C12 WGBS (Wilcoxon test, paired), * P = 0.023 for CTCF M-ChIP versus WGBS (logistic regression model). c Scatter plot showing the relationship between binding strength and CpG methylation within the KLF4 M-ChIP peaks (Pearson correlation = 0.25; bottom left corner: 5138 CpGs, top left corner: 578 CpGs, top right corner: 104 CpGs, bottom right corner: 60 CpGs). d Venn diagram showing the overlap between WT and mutant KLF4 M-ChIP peaks. e Top: Illustration of KLF4 motifs from the Jaspar database (MA0039.1 and MA0039.2). The black bar represents the potential CpGs present in the MA0039.2 motif. Bottom: histogram showing the relative distribution of KLF4 motifs in WT, mutant and common KLF4 M-ChIP peaks using FIMO from the MEME suite. Absolute numbers of each motif are indicated. f Heatmap showing M-ATAC signal intensity at KLF4 M-ChIP peaks that are specific to WT (1836 peaks), mutant (267 peaks), or common between both conditions (303 peaks). g Average cytosine methylation from M-ATAC in WT versus mutant KLF4 expressing cells in WT specific KLF4 M-ChIP peaks (Pearson correlation = 0.78, p value
    Figure Legend Snippet: M-ChIP enables analysis of DNA methylation binding by CTCF and KLF4. a Top: Schematic illustration representing an ATAC-seq peak with a CTCF motif and CTCF occupancy dependent on C2 and C12 methylation. Bottom: average profiles of M-ATAC (left) and CTCF M-ChIP (right) intensity at CpGs in a CTCF motif within M-ATAC peaks for the four groups of CpGs (group #1: 288 CpGs, group #2: 17133 CpGs, group #3 CpGs: 758, group #4: 25 CpGs). b top: CTCF motif from JASPAR database (MA0139.1). The 2 key CpG positions (C2 and C12) are indicated. Bottom: violin plots of methylation percentage from CTCF M-ChIP and WGBS, at C2 and C12 positions in the CTCF motif (MA0139.1). *** P = 1.02e−12 for C2 CTCF M-ChIP versus C12 CTCF M-ChIP (Wilcoxon test), ** P = 0.008 for C2 WGBS versus C12 WGBS (Wilcoxon test), *** P = 9e−12 for C2 CTCF M-ChIP versus C2 WGBS (Wilcoxon test, paired), *** P = 0.00075 for C12 CTCF M-ChIP versus C12 WGBS (Wilcoxon test, paired), * P = 0.023 for CTCF M-ChIP versus WGBS (logistic regression model). c Scatter plot showing the relationship between binding strength and CpG methylation within the KLF4 M-ChIP peaks (Pearson correlation = 0.25; bottom left corner: 5138 CpGs, top left corner: 578 CpGs, top right corner: 104 CpGs, bottom right corner: 60 CpGs). d Venn diagram showing the overlap between WT and mutant KLF4 M-ChIP peaks. e Top: Illustration of KLF4 motifs from the Jaspar database (MA0039.1 and MA0039.2). The black bar represents the potential CpGs present in the MA0039.2 motif. Bottom: histogram showing the relative distribution of KLF4 motifs in WT, mutant and common KLF4 M-ChIP peaks using FIMO from the MEME suite. Absolute numbers of each motif are indicated. f Heatmap showing M-ATAC signal intensity at KLF4 M-ChIP peaks that are specific to WT (1836 peaks), mutant (267 peaks), or common between both conditions (303 peaks). g Average cytosine methylation from M-ATAC in WT versus mutant KLF4 expressing cells in WT specific KLF4 M-ChIP peaks (Pearson correlation = 0.78, p value

    Techniques Used: Chromatin Immunoprecipitation, DNA Methylation Assay, Binding Assay, Methylation, CpG Methylation Assay, Mutagenesis, Expressing

    7) Product Images from "Primary 1,25-Dihydroxyvitamin D3 Response of the Interleukin 8 Gene Cluster in Human Monocyte- and Macrophage-Like Cells"

    Article Title: Primary 1,25-Dihydroxyvitamin D3 Response of the Interleukin 8 Gene Cluster in Human Monocyte- and Macrophage-Like Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078170

    Genome view of the CXCL gene cluster. A. The IGV browser was used to show the peak tracks of FAIRE-seq data from THP-1 cells [ 51 ] (stimulated for 20 min with ethanol, turquoise) and VDR ChIP-seq data from THP-1 cells [ 32 ] (unstimulated (-) and treated for 40 min with 1,25(OH) 2 D 3 (+), red). The gene structures are shown in blue and the 9 genes of the CXCL gene cluster are underlayed in grey. The THP-1 data were compared with CTCF ChIP-seq data from the ENCODE cell lines K562, HUVEC and NHEK [ 42 ] (blue) and CTCF ChIA-PET data [ 47 ] in track view (dark blue) and in looping view (grey horizontal lines). Six conserved CTCF sites were highlighted. B. ChIP-qPCR was performed with chromatin samples obtained from unstimulated THP-1 cells to determine CTCF (blue) and unspecific IgG (grey) binding at the six genomic regions, which were suggested by data obtained in K562 cells (see panel A). Columns represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance CTCF association in reference to a control region from chromosome 6 (* p
    Figure Legend Snippet: Genome view of the CXCL gene cluster. A. The IGV browser was used to show the peak tracks of FAIRE-seq data from THP-1 cells [ 51 ] (stimulated for 20 min with ethanol, turquoise) and VDR ChIP-seq data from THP-1 cells [ 32 ] (unstimulated (-) and treated for 40 min with 1,25(OH) 2 D 3 (+), red). The gene structures are shown in blue and the 9 genes of the CXCL gene cluster are underlayed in grey. The THP-1 data were compared with CTCF ChIP-seq data from the ENCODE cell lines K562, HUVEC and NHEK [ 42 ] (blue) and CTCF ChIA-PET data [ 47 ] in track view (dark blue) and in looping view (grey horizontal lines). Six conserved CTCF sites were highlighted. B. ChIP-qPCR was performed with chromatin samples obtained from unstimulated THP-1 cells to determine CTCF (blue) and unspecific IgG (grey) binding at the six genomic regions, which were suggested by data obtained in K562 cells (see panel A). Columns represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance CTCF association in reference to a control region from chromosome 6 (* p

    Techniques Used: Chromatin Immunoprecipitation, ChIA Pet Assay, Real-time Polymerase Chain Reaction, Binding Assay, Two Tailed Test

    Related Articles

    Protease Inhibitor:

    Article Title: The Dynamic Chromatin Architecture of the Regenerating Liver
    Article Snippet: .. A total of 10 μg sheared DNA was incubated with anti-CTCF (2 μg, 07-729; Millipore, Burlington, MA) or anti-HNF4α (2 μg, ab181604; Abcam) antibodies in dilution buffer (16.7 mmol/L Tris-HCl pH 8.1, 167 mmol/L NaCl, 0.01% SDS, 1.1% Triton-X 100, and protease inhibitor) at 4°C overnight. .. Protein A agarose beads also were washed with cold dilution buffer 3 times and incubated with blocking buffer (10 mg/mL bovine serum albumin, ChIP dilution buffer, and protease inhibitor) at 4°C overnight.

    Immunoprecipitation:

    Article Title: Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells
    Article Snippet: .. Protein immunoprecipitation (IP)CTCF IP was performed in 226LDM cells, using anti-CTCF antibody [ ]. .. 226LDM cells cultured in a T75 flask were trypsinized, washed twice with PBS and then lysed by vortexing in BF2 (25 mM Tris/Hepes - pH 8.0, 2 mM EDTA, 0.5% Tween20, 0.5 M NaCl, 1:100 Halt protease inhibitor cocktail).

    Incubation:

    Article Title: CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells
    Article Snippet: .. The chromatin was precleared using protein A/G-agarose (Upstate) and incubated with anti-CTCF antibody (07-729; Millipore), anti-RAD21 antibody (ab992; Abcam), anti-H3K27me3 antibody (07-449; Millipore), anti-EZH2 antibody (E80420_612666; BD), or rabbit/mouse IgG (as the control) at 4°C overnight. .. The immunoprecipitates were recovered by incubation with protein A/G-agarose (Upstate) at 4°C for 2 h, followed by a low-speed centrifugation step, and the washed pellets were reverse cross-linked.

    Article Title: CTCF Binding to the First Intron of the Major Immediate Early (MIE) Gene of Human Cytomegalovirus (HCMV) Negatively Regulates MIE Gene Expression and HCMV Replication
    Article Snippet: .. The anti-CTCF antibody or normal IgG was incubated with the DNA-protein mixture, and ChIP assays were performed with the EZChIP kit (Millipore, Billerica, MA) according to the manufacturer's instructions. ..

    Article Title: Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation
    Article Snippet: .. Membrane was incubated overnight with anti-Smchd1 antibody (1:2,000 diluted, A302-871A; Bethyl), anti-Ctcf antibody (1:2,000 diluted, 07-729; Millipore), and antitubulin antibody (1:2,000 diluted, sc-23948; Santa Cruz Biotechnology), respectively, at 4 °C, followed with HRP-conjugated secondary antibodies. .. Membrane was visualized using an ECL system (Luminata; Millipore) following the manufacturer’s instructions.

    Article Title: The Dynamic Chromatin Architecture of the Regenerating Liver
    Article Snippet: .. A total of 10 μg sheared DNA was incubated with anti-CTCF (2 μg, 07-729; Millipore, Burlington, MA) or anti-HNF4α (2 μg, ab181604; Abcam) antibodies in dilution buffer (16.7 mmol/L Tris-HCl pH 8.1, 167 mmol/L NaCl, 0.01% SDS, 1.1% Triton-X 100, and protease inhibitor) at 4°C overnight. .. Protein A agarose beads also were washed with cold dilution buffer 3 times and incubated with blocking buffer (10 mg/mL bovine serum albumin, ChIP dilution buffer, and protease inhibitor) at 4°C overnight.

    Western Blot:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody. .. 293T cells were used as an expression system for the Flag-tagged proteins, as F9 cells transfect with a much lower relative efficiency.

    Article Title: The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress
    Article Snippet: .. Antibodies Antibodies used for western blot analysis were rabbit anti-CSB (1:2000) , rabbit anti-CTCF (1:2000) (Millipore, 07-729), mouse anti-GAPDH (1:10 000) (Millipore, MAB374), rabbit anti-BRG1 (1:1000) , rabbit anti-acetylated histone H3 (1:1000) (Millipore, 06-599), HRP-conjugated goat anti-rabbit IgG (1:10 000) (Pierce, 31460) and HRP-conjugated goat anti-mouse (IgG+IgM) (1:10 000) (Jackson Laboratory, 115-035-044). ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat07-729) or the rabbit anti-RBPJ antibody. .. 293T cells were used as an expression system for the Flag-tagged proteins, as F9 cells transfect with a much lower relative efficiency.

    Chromatin Immunoprecipitation:

    Article Title: CTCF Binding to the First Intron of the Major Immediate Early (MIE) Gene of Human Cytomegalovirus (HCMV) Negatively Regulates MIE Gene Expression and HCMV Replication
    Article Snippet: .. The anti-CTCF antibody or normal IgG was incubated with the DNA-protein mixture, and ChIP assays were performed with the EZChIP kit (Millipore, Billerica, MA) according to the manufacturer's instructions. ..

    Article Title: Control of directionality of chromatin folding for the inter- and intra-domain contacts at the Tfap2c–Bmp7 locus
    Article Snippet: .. Three independent ChIP experiments were performed for both anti-CTCF antibody and IgG control. ..

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    Millipore anti ctcf antibody
    A schematic model illustrating the events observed in control and treated <t>226LDM</t> cells in which transition from CTCF130 to CTCF180 takes place. Following treatment, cells change morphologically from adherent and flat to suspended and rounded. PARylated CTCF180 in treated cells is largely redistributed from the cell nucleus into cytoplasm (depicted on top of the Figure). More GC-rich stronger common sites retain CTCF180 (with smaller strengths). CTCF180 is evacuated from weaker (lost) sites. Gained sites characterised by the absence of the <t>CTCF</t> motif acquire CTCF180 after treatment possibly due to interaction with additional proteins or may be just false positives. Nucleosome occupancy associated with the higher levels of the H3K9me3 is increased in the regions overlapping with CTCF sites in common and lost groups. Molecular changes within regions containing these CTCF sites result in alterations in gene expression patterns.
    Anti Ctcf Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A schematic model illustrating the events observed in control and treated 226LDM cells in which transition from CTCF130 to CTCF180 takes place. Following treatment, cells change morphologically from adherent and flat to suspended and rounded. PARylated CTCF180 in treated cells is largely redistributed from the cell nucleus into cytoplasm (depicted on top of the Figure). More GC-rich stronger common sites retain CTCF180 (with smaller strengths). CTCF180 is evacuated from weaker (lost) sites. Gained sites characterised by the absence of the CTCF motif acquire CTCF180 after treatment possibly due to interaction with additional proteins or may be just false positives. Nucleosome occupancy associated with the higher levels of the H3K9me3 is increased in the regions overlapping with CTCF sites in common and lost groups. Molecular changes within regions containing these CTCF sites result in alterations in gene expression patterns.

    Journal: bioRxiv

    Article Title: Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

    doi: 10.1101/175448

    Figure Lengend Snippet: A schematic model illustrating the events observed in control and treated 226LDM cells in which transition from CTCF130 to CTCF180 takes place. Following treatment, cells change morphologically from adherent and flat to suspended and rounded. PARylated CTCF180 in treated cells is largely redistributed from the cell nucleus into cytoplasm (depicted on top of the Figure). More GC-rich stronger common sites retain CTCF180 (with smaller strengths). CTCF180 is evacuated from weaker (lost) sites. Gained sites characterised by the absence of the CTCF motif acquire CTCF180 after treatment possibly due to interaction with additional proteins or may be just false positives. Nucleosome occupancy associated with the higher levels of the H3K9me3 is increased in the regions overlapping with CTCF sites in common and lost groups. Molecular changes within regions containing these CTCF sites result in alterations in gene expression patterns.

    Article Snippet: Protein immunoprecipitation (IP)CTCF IP was performed in 226LDM cells, using anti-CTCF antibody [ ].

    Techniques: Expressing

    Western Blot analyses of primary breast tissues and breast cell lines demonstrating the presence of two forms, CTF130 and CTCF180, with the antibodies prescreened as previously described [ 1 ]. A, B and C: CTCF migrates as 180 kDa protein in normal breast tissues, CTCF-130 appears in tumour breast tissues, and both forms of CTCF are present in MCF-7 breast cancer cells and 226LDM immortalized breast cells. A. Western blot analysis of three independent paired samples of normal and tumour tissues (“a”, “b” and “c”). Tissue lysates (50 μg of the total protein) prepared as previously described [5] were resolved by SDS-PAGE, blotted and probed with the pre-screened anti-CTCF polyclonal antibody recognising CTCF180 and CTCF130 (“CTCF 130/180”, upper panel), stripped and re-probed with the anti-CTCF antibody recognising only CTCF130 (“CTCF130”, middle panel), re-stripped and re-probed with the anti-tubulin antibody (“Tubulin”, lower panel). B. Western blot analysis of two independent paired samples of normal (N) and tumour (T) tissues (“d” and “f”) together with the lysate from breast cancer cell line MCF7 (far right) performed as described above. C. Western blot analysis of lysate from breast cancer cell line MCF7 and immortalized breast cells 226LDM probed with the pre-screened anti-CTCF polyclonal antibody recognising CTCF180 and CTCF130. D. Western blot analysis of lysates from control and hydroxyurea/nocodazole treated 226LDM cells 226LDM cells were cell-cycle arrested by addition of 100mM hydroxyurea and 500ng/ml nocodazole in their culture medium (as described in the Supplementary materials and methods section) probed with the pre-screened anti-CTCF polyclonal antibody recognising CTCF180 and CTCF130. The development of the membranes was performed with the UptiLight™ chemiluminescence substrate. Tubulin was used as a loading control. Positions of CTCF-180, CTCF-130and tubulin are indicated. “N”- and “T” refer to normal and tumour breast tissues. E. Quantification of the gel from panel D performed using ImageJ [ 2 ] following the standard instructions provided by the developers. In particular, the intensities of all bands were normalised by that of the corresponding tubulin band, and then normalised by the intensity of the band of CTCF130 in control. Following normalisation the value of CTCF130 in control was designated as 1.

    Journal: bioRxiv

    Article Title: Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

    doi: 10.1101/175448

    Figure Lengend Snippet: Western Blot analyses of primary breast tissues and breast cell lines demonstrating the presence of two forms, CTF130 and CTCF180, with the antibodies prescreened as previously described [ 1 ]. A, B and C: CTCF migrates as 180 kDa protein in normal breast tissues, CTCF-130 appears in tumour breast tissues, and both forms of CTCF are present in MCF-7 breast cancer cells and 226LDM immortalized breast cells. A. Western blot analysis of three independent paired samples of normal and tumour tissues (“a”, “b” and “c”). Tissue lysates (50 μg of the total protein) prepared as previously described [5] were resolved by SDS-PAGE, blotted and probed with the pre-screened anti-CTCF polyclonal antibody recognising CTCF180 and CTCF130 (“CTCF 130/180”, upper panel), stripped and re-probed with the anti-CTCF antibody recognising only CTCF130 (“CTCF130”, middle panel), re-stripped and re-probed with the anti-tubulin antibody (“Tubulin”, lower panel). B. Western blot analysis of two independent paired samples of normal (N) and tumour (T) tissues (“d” and “f”) together with the lysate from breast cancer cell line MCF7 (far right) performed as described above. C. Western blot analysis of lysate from breast cancer cell line MCF7 and immortalized breast cells 226LDM probed with the pre-screened anti-CTCF polyclonal antibody recognising CTCF180 and CTCF130. D. Western blot analysis of lysates from control and hydroxyurea/nocodazole treated 226LDM cells 226LDM cells were cell-cycle arrested by addition of 100mM hydroxyurea and 500ng/ml nocodazole in their culture medium (as described in the Supplementary materials and methods section) probed with the pre-screened anti-CTCF polyclonal antibody recognising CTCF180 and CTCF130. The development of the membranes was performed with the UptiLight™ chemiluminescence substrate. Tubulin was used as a loading control. Positions of CTCF-180, CTCF-130and tubulin are indicated. “N”- and “T” refer to normal and tumour breast tissues. E. Quantification of the gel from panel D performed using ImageJ [ 2 ] following the standard instructions provided by the developers. In particular, the intensities of all bands were normalised by that of the corresponding tubulin band, and then normalised by the intensity of the band of CTCF130 in control. Following normalisation the value of CTCF130 in control was designated as 1.

    Article Snippet: Protein immunoprecipitation (IP)CTCF IP was performed in 226LDM cells, using anti-CTCF antibody [ ].

    Techniques: Western Blot, SDS Page

    Immunoprecipitation of CTCF in 226LDM cells with the anti-CTCF polyclonal antibody: Western blot analysis. Protein extracts from untreated 226LDM cells were used in a series of protein immunoprecipitation experiments to confirm that both forms of CTCF (CTCF130 and CTCF180) can be immunoprecipitated with the selected anti-CTCF antibody (experimental details are described in the “Supplemental Materials and Methods” section). The proteins were resolved by SDS-PAGE, blotted, and probed with the selected anti-CTCF antibody. The visualization of the signal was performed using UptiLight™. Arrows indicate the positions of the two CTCF forms. Keys: Input: Pre-cleared and pre-blocked extracts (20 μl) from 226LDM cells used for the immunoprecipitation experiments. IP: Immunoprecipitated proteins (5 μl) from lysed sepharose beads. Sup: Supernatant material (20 μl) collected after centrifugation of beads with the immunoprecipitated proteins. Wash: Material (20 μl) from the wash with the immunoprecipitation buffer (BF1+BF2). No antibody: samples from the experiments performed using the same methods but without the selected CTCF antibody (used as control for the experiment.) No antibody IP: Proteins (5 μl) from lysed sepharose beads. Sup: Supernatant material (20 μl) collected after centrifugation of beads with the pre-cleared and pre-blocked extracts protein extracts.

    Journal: bioRxiv

    Article Title: Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

    doi: 10.1101/175448

    Figure Lengend Snippet: Immunoprecipitation of CTCF in 226LDM cells with the anti-CTCF polyclonal antibody: Western blot analysis. Protein extracts from untreated 226LDM cells were used in a series of protein immunoprecipitation experiments to confirm that both forms of CTCF (CTCF130 and CTCF180) can be immunoprecipitated with the selected anti-CTCF antibody (experimental details are described in the “Supplemental Materials and Methods” section). The proteins were resolved by SDS-PAGE, blotted, and probed with the selected anti-CTCF antibody. The visualization of the signal was performed using UptiLight™. Arrows indicate the positions of the two CTCF forms. Keys: Input: Pre-cleared and pre-blocked extracts (20 μl) from 226LDM cells used for the immunoprecipitation experiments. IP: Immunoprecipitated proteins (5 μl) from lysed sepharose beads. Sup: Supernatant material (20 μl) collected after centrifugation of beads with the immunoprecipitated proteins. Wash: Material (20 μl) from the wash with the immunoprecipitation buffer (BF1+BF2). No antibody: samples from the experiments performed using the same methods but without the selected CTCF antibody (used as control for the experiment.) No antibody IP: Proteins (5 μl) from lysed sepharose beads. Sup: Supernatant material (20 μl) collected after centrifugation of beads with the pre-cleared and pre-blocked extracts protein extracts.

    Article Snippet: Protein immunoprecipitation (IP)CTCF IP was performed in 226LDM cells, using anti-CTCF antibody [ ].

    Techniques: Immunoprecipitation, Western Blot, SDS Page, Centrifugation

    Localization profile of CTCF in control and treated 226LDM cells. A) CTCF immunofluorescence staining of control (untreated) and treated 226LDM cells using the polyclonal anti-CTCF antibody that recognizes both CTCF130 and CTCF180. In control cells the CTCF signal (green) appears predominantly diffused in the nuclear area. In treated cells staining is mostly detected in the cytoplasm. Blue colour shows DAPI staining of the nucleus. B) Average CTCF immunofluorescence (IF) signal intensity ratio between the nucleus and the whole cell calculated separately for the treatment and control conditions. The quantification was performed using ImageJ [ 2 ] using three individual cells (N=3) on the same slide. All ratios of intensities were normalised by that in the control. The box height shows the standard deviation calculated based on three independent measurements (three cells). The horizontal line inside the box shows the median values. Keys: “CTCF-FITC” – Staining of CTCF and with secondary antibodies conjugated with a fluorescent dyes, (e.g. FITC. “DAPI” – visualisation of nuclei with the DNA binding dye, DAPI (4’,6-diamidino-2-phenylindole, dilactate). “Merge” – overlay of CTCF-FITC and DAPI fluorescent images.

    Journal: bioRxiv

    Article Title: Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

    doi: 10.1101/175448

    Figure Lengend Snippet: Localization profile of CTCF in control and treated 226LDM cells. A) CTCF immunofluorescence staining of control (untreated) and treated 226LDM cells using the polyclonal anti-CTCF antibody that recognizes both CTCF130 and CTCF180. In control cells the CTCF signal (green) appears predominantly diffused in the nuclear area. In treated cells staining is mostly detected in the cytoplasm. Blue colour shows DAPI staining of the nucleus. B) Average CTCF immunofluorescence (IF) signal intensity ratio between the nucleus and the whole cell calculated separately for the treatment and control conditions. The quantification was performed using ImageJ [ 2 ] using three individual cells (N=3) on the same slide. All ratios of intensities were normalised by that in the control. The box height shows the standard deviation calculated based on three independent measurements (three cells). The horizontal line inside the box shows the median values. Keys: “CTCF-FITC” – Staining of CTCF and with secondary antibodies conjugated with a fluorescent dyes, (e.g. FITC. “DAPI” – visualisation of nuclei with the DNA binding dye, DAPI (4’,6-diamidino-2-phenylindole, dilactate). “Merge” – overlay of CTCF-FITC and DAPI fluorescent images.

    Article Snippet: Protein immunoprecipitation (IP)CTCF IP was performed in 226LDM cells, using anti-CTCF antibody [ ].

    Techniques: Immunofluorescence, Staining, Standard Deviation, Binding Assay

    Analysis of CTCF binding and gene expression profiles in control and treated 226LDM cells. (A) Schematic illustration of three groups of CTCF sites detected by ChIP-seq in control and treated cells: common sites are present in both cell states, lost sites are present only in control cells, and gained sites appear only in treated cells. (B) A pie chart showing the numbers of common, lost and gained CTCF sites. (C) Association of gene expression patterns in control and treated cells with the three groups of CTCF binding sites present within +/− 10,000 bp from TSS. Numbers of genes up-regulated, down-regulated and unchanged is shown for all genes near CTCF according to the above criterium (left). Percentages and numbers of genes with different expression patterns for each group of CTCF-binding sites are shown in the middle and right panels, respectively. (D) Association of gene expression patterns in control and treated cells with the three groups of CTCF binding sites present within +/− 10,000 bp from TSS of housekeeping genes. Numbers of genes up-regulated, down-regulated and unchanged is shown for all genes near CTCF (left). Percentages and numbers of genes with different expression patterns for each group of CTCF-binding sites are shown in the right panel. (E) Gene ontology terms enriched for genes containinig CTCF within +/−10,000bp from TSS. Genes are ordered by expression fold change. Red colour corresponds to up-regulation, green colour – down-regulation.

    Journal: bioRxiv

    Article Title: Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

    doi: 10.1101/175448

    Figure Lengend Snippet: Analysis of CTCF binding and gene expression profiles in control and treated 226LDM cells. (A) Schematic illustration of three groups of CTCF sites detected by ChIP-seq in control and treated cells: common sites are present in both cell states, lost sites are present only in control cells, and gained sites appear only in treated cells. (B) A pie chart showing the numbers of common, lost and gained CTCF sites. (C) Association of gene expression patterns in control and treated cells with the three groups of CTCF binding sites present within +/− 10,000 bp from TSS. Numbers of genes up-regulated, down-regulated and unchanged is shown for all genes near CTCF according to the above criterium (left). Percentages and numbers of genes with different expression patterns for each group of CTCF-binding sites are shown in the middle and right panels, respectively. (D) Association of gene expression patterns in control and treated cells with the three groups of CTCF binding sites present within +/− 10,000 bp from TSS of housekeeping genes. Numbers of genes up-regulated, down-regulated and unchanged is shown for all genes near CTCF (left). Percentages and numbers of genes with different expression patterns for each group of CTCF-binding sites are shown in the right panel. (E) Gene ontology terms enriched for genes containinig CTCF within +/−10,000bp from TSS. Genes are ordered by expression fold change. Red colour corresponds to up-regulation, green colour – down-regulation.

    Article Snippet: Protein immunoprecipitation (IP)CTCF IP was performed in 226LDM cells, using anti-CTCF antibody [ ].

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation

    CSB regulates a subset of CTCF occupancy sites upon oxidative stress. ( A ) CTCF ChIP-qPCR assays in CSB expressing (WT) and non-expressing (CS1AN) cells, with or without a 1-h menadione treatment (100 μM). Shown are means ± SEM ( n = 3). A paired t -test was used to determine if the difference in CTCF enrichment before and after menadione treatment was significant. Single asterisks indicate P values

    Journal: Nucleic Acids Research

    Article Title: The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress

    doi: 10.1093/nar/gkv1219

    Figure Lengend Snippet: CSB regulates a subset of CTCF occupancy sites upon oxidative stress. ( A ) CTCF ChIP-qPCR assays in CSB expressing (WT) and non-expressing (CS1AN) cells, with or without a 1-h menadione treatment (100 μM). Shown are means ± SEM ( n = 3). A paired t -test was used to determine if the difference in CTCF enrichment before and after menadione treatment was significant. Single asterisks indicate P values

    Article Snippet: Antibodies Antibodies used for western blot analysis were rabbit anti-CSB (1:2000) , rabbit anti-CTCF (1:2000) (Millipore, 07-729), mouse anti-GAPDH (1:10 000) (Millipore, MAB374), rabbit anti-BRG1 (1:1000) , rabbit anti-acetylated histone H3 (1:1000) (Millipore, 06-599), HRP-conjugated goat anti-rabbit IgG (1:10 000) (Pierce, 31460) and HRP-conjugated goat anti-mouse (IgG+IgM) (1:10 000) (Jackson Laboratory, 115-035-044).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

    CTCF collaborates with CSB in response to oxidative stress. ( A ) Motif analysis of CSB ChIP-seq data. ( B ) Menadione sensitivity assays on CSB expressing and non-expressing (Vector) cells with decreased CTCF levels. Shown are means ± SEM ( n = 4). ( C ) Western blot showing a reduction in the CTCF protein level in cells expressing CTCF shRNA. Relative CTCF levels are shown below the CTCF blot. ( D ) CSB ChIP-qPCR assays in cells infected with lentivirus expressing control or CTCF shRNA, with or without with a 1-h menadione treatment (100 μM). Shown are means ± SEM ( n = 3). A paired t -test was used to determine if the difference in CSB enrichment with and without CTCF shRNA treatment was significant. Asterisks indicate P -values

    Journal: Nucleic Acids Research

    Article Title: The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress

    doi: 10.1093/nar/gkv1219

    Figure Lengend Snippet: CTCF collaborates with CSB in response to oxidative stress. ( A ) Motif analysis of CSB ChIP-seq data. ( B ) Menadione sensitivity assays on CSB expressing and non-expressing (Vector) cells with decreased CTCF levels. Shown are means ± SEM ( n = 4). ( C ) Western blot showing a reduction in the CTCF protein level in cells expressing CTCF shRNA. Relative CTCF levels are shown below the CTCF blot. ( D ) CSB ChIP-qPCR assays in cells infected with lentivirus expressing control or CTCF shRNA, with or without with a 1-h menadione treatment (100 μM). Shown are means ± SEM ( n = 3). A paired t -test was used to determine if the difference in CSB enrichment with and without CTCF shRNA treatment was significant. Asterisks indicate P -values

    Article Snippet: Antibodies Antibodies used for western blot analysis were rabbit anti-CSB (1:2000) , rabbit anti-CTCF (1:2000) (Millipore, 07-729), mouse anti-GAPDH (1:10 000) (Millipore, MAB374), rabbit anti-BRG1 (1:1000) , rabbit anti-acetylated histone H3 (1:1000) (Millipore, 06-599), HRP-conjugated goat anti-rabbit IgG (1:10 000) (Pierce, 31460) and HRP-conjugated goat anti-mouse (IgG+IgM) (1:10 000) (Jackson Laboratory, 115-035-044).

    Techniques: Chromatin Immunoprecipitation, Expressing, Plasmid Preparation, Western Blot, shRNA, Real-time Polymerase Chain Reaction, Infection

    CSB interacts with CTCF in cells and in vitro . ( A ) Co-immunoprecipitation of CSB and CTCF in 293T transiently transfected with Flag-tagged CTCF, with or without a 1-h treatment of 100 μM menadione. 3.3% of the lysates used for IP were loaded as input. ( B ) Schematics of recombinant proteins used in ( C – E ). All CSB derivatives were N-terminally tagged with the Flag epitope. (C) Coomassie-stained gel showing that CSB directly interacts with CTCF. CSB-C, but not CSB-N, is sufficient for the CTCF association. MBP was used as a negative control. (D and E) EMSA assays showing that CSB enhances CTCF association with DNA. (D) Varying amounts of purified MBP-CTCF (lane 2 in C) or MBP (lane 1 in C) were incubated with a 32 P-labeled, 200 bp DNA fragment containing a CTCF-binding motif (Supplementary Figure S4B). Protein–DNA complexes were resolved in a native 5% polyacrylamide gel. (E) Varying amounts of purified CSB were incubated with the radiolabeled DNA fragment in the presence or absence of MBP-CTCF. Reactions were subsequently resolved in a 5% native polyacrylamide gel. Protein–DNA complexes marked by ‘•’ and ‘••’ contain the MBP-CTCF protein, as they interacted with an anti-MBP antibody (Supplementary Figure S4A).

    Journal: Nucleic Acids Research

    Article Title: The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress

    doi: 10.1093/nar/gkv1219

    Figure Lengend Snippet: CSB interacts with CTCF in cells and in vitro . ( A ) Co-immunoprecipitation of CSB and CTCF in 293T transiently transfected with Flag-tagged CTCF, with or without a 1-h treatment of 100 μM menadione. 3.3% of the lysates used for IP were loaded as input. ( B ) Schematics of recombinant proteins used in ( C – E ). All CSB derivatives were N-terminally tagged with the Flag epitope. (C) Coomassie-stained gel showing that CSB directly interacts with CTCF. CSB-C, but not CSB-N, is sufficient for the CTCF association. MBP was used as a negative control. (D and E) EMSA assays showing that CSB enhances CTCF association with DNA. (D) Varying amounts of purified MBP-CTCF (lane 2 in C) or MBP (lane 1 in C) were incubated with a 32 P-labeled, 200 bp DNA fragment containing a CTCF-binding motif (Supplementary Figure S4B). Protein–DNA complexes were resolved in a native 5% polyacrylamide gel. (E) Varying amounts of purified CSB were incubated with the radiolabeled DNA fragment in the presence or absence of MBP-CTCF. Reactions were subsequently resolved in a 5% native polyacrylamide gel. Protein–DNA complexes marked by ‘•’ and ‘••’ contain the MBP-CTCF protein, as they interacted with an anti-MBP antibody (Supplementary Figure S4A).

    Article Snippet: Antibodies Antibodies used for western blot analysis were rabbit anti-CSB (1:2000) , rabbit anti-CTCF (1:2000) (Millipore, 07-729), mouse anti-GAPDH (1:10 000) (Millipore, MAB374), rabbit anti-BRG1 (1:1000) , rabbit anti-acetylated histone H3 (1:1000) (Millipore, 06-599), HRP-conjugated goat anti-rabbit IgG (1:10 000) (Pierce, 31460) and HRP-conjugated goat anti-mouse (IgG+IgM) (1:10 000) (Jackson Laboratory, 115-035-044).

    Techniques: In Vitro, Immunoprecipitation, Transfection, Recombinant, FLAG-tag, Staining, Negative Control, Purification, Incubation, Labeling, Binding Assay

    Deletion of the CTCF motif from intron A led to increases in HCMV IE gene expression and viral DNA replication. (A) ChIP-qPCR analysis of CTCF or control IgG with primers specific for the regions indicated in MRC-5 cells infected with HCMVwt, HCMVdCTCFiRev,

    Journal: Journal of Virology

    Article Title: CTCF Binding to the First Intron of the Major Immediate Early (MIE) Gene of Human Cytomegalovirus (HCMV) Negatively Regulates MIE Gene Expression and HCMV Replication

    doi: 10.1128/JVI.00845-14

    Figure Lengend Snippet: Deletion of the CTCF motif from intron A led to increases in HCMV IE gene expression and viral DNA replication. (A) ChIP-qPCR analysis of CTCF or control IgG with primers specific for the regions indicated in MRC-5 cells infected with HCMVwt, HCMVdCTCFiRev,

    Article Snippet: The anti-CTCF antibody or normal IgG was incubated with the DNA-protein mixture, and ChIP assays were performed with the EZChIP kit (Millipore, Billerica, MA) according to the manufacturer's instructions.

    Techniques: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Infection

    The effect of CTCF knockdown on H3K27me3 modification and EZH2 enrichment at the HOXA locus in NT2/D1 cells with and without RA induction. (A and B) H3K27me3 enrichment at the HOXA locus in RiGFP- and RiCTCF-treated NT2/D1 cells before (A) and after (B)

    Journal: Molecular and Cellular Biology

    Article Title: CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells

    doi: 10.1128/MCB.00567-14

    Figure Lengend Snippet: The effect of CTCF knockdown on H3K27me3 modification and EZH2 enrichment at the HOXA locus in NT2/D1 cells with and without RA induction. (A and B) H3K27me3 enrichment at the HOXA locus in RiGFP- and RiCTCF-treated NT2/D1 cells before (A) and after (B)

    Article Snippet: The chromatin was precleared using protein A/G-agarose (Upstate) and incubated with anti-CTCF antibody (07-729; Millipore), anti-RAD21 antibody (ab992; Abcam), anti-H3K27me3 antibody (07-449; Millipore), anti-EZH2 antibody (E80420_612666; BD), or rabbit/mouse IgG (as the control) at 4°C overnight.

    Techniques: Modification