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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    Structured Review

    Millipore ctcf antibody
    Anti CTCF antibody

    https://www.bioz.com/result/ctcf antibody/product/Millipore
    Average 95 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    ctcf antibody - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    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 "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

    8) 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

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    Centrifugation:

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    Article Title: CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells
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    Article Title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination
    Article Snippet: High-salt extraction of nuclear proteins was carried out on ice for 30 min in 150 μL buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM DTT and 1 mM PMSF) followed by centrifugation (17,000 g at 4°C for 10 min). .. Typically, 10 μg of whole-cell and 20 μg of nuclear protein extracts were used for immunoblot with anti-DDX1 (NovusBiologicals or ProteinTech), anti-AID (Cell Signaling L7E7), anti-Tubulin (Sigma-Aldrich), anti-Actin (Sigma-Aldrich), anti-CTCF (Millipore) or anti-Histone H3 (Abcam) and HRP-conjugated secondary antibodies in TBST, 5% milk (Sigma-Aldrich).

    Immunocytochemistry:

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    Article Snippet: Paragraph title: Immunocytochemistry ... Anti-BDNF (1:300; Santa Cruz Biotechnology, CA, USA) and anti-CTCF (1:300, Millipore, MA, USA) antibodies, diluted in 1% bovine serum albumin (BSA)/TBST were added to the cells and incubated overnight at 4°C.

    Construct:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: Co-IP, ChIP and real-time PCR experiments pCDNA3 constructs expressing Flag-tagged CTCF or Flag-tagged RBPJ were transfected into 293T cells. .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    Real-time Polymerase Chain Reaction:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: Paragraph title: Co-IP, ChIP and real-time PCR experiments ... Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    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 DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated with ethanol, and used for real-time PCR analysis.

    Article Title: Higher-Order Chromatin Regulation and Differential Gene Expression in the Human Tumor Necrosis Factor/Lymphotoxin Locus in Hepatocellular Carcinoma Cells
    Article Snippet: .. In order to clarify the localization of CTCF and the cofactor cohesin RAD21 in hepatic cells, we performed chromatin immunoprecipitation (ChIP) analyses using anti-CTCF and anti-RAD21 antibodies, followed by quantitative PCR (qPCR) ( C and D). ..

    Incubation:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: The beads were then washed four times with PBS/0.05% Triton X-100 and immobilized Flag-tagged CTCF or Flag-tagged RBPJ were mixed with F9 cell lysates, prepared as described above, and incubated overnight. .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    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: Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology
    Article Snippet: For immunoprecipitation, the indicated antibodies were added to pre-cleared lysates along with 40 µl of a 50% slurry of 1∶1 protein A/G beads and incubated at 4°C overnight. .. Antibodies used were: α-CTCF (Millipore), α-Rad21 (Abcam), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz) and α-PAR (Millipore).

    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: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: .. Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads. .. After washing and elution, DNA was incubated at 65°C overnight for reverse cross-linking, followed by RNase A, proteinase K treatment and DNA precipitation.

    Article Title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination
    Article Snippet: For nuclear protein extracts, 10x106 cells were resuspended in 500 μL cell lysis buffer (50 mM Tris-HCl, pH 8, 300 mM Sucrose, 4 mM MgAc, 12.5 mM KCl, 1 mM DTT, 10 mM β-mercaptoethanol, 0.3% NP-40 and 1mM PMSF) and incubated 5 min on ice followed by centrifugation (500 g for 10 min at 4°C). .. Typically, 10 μg of whole-cell and 20 μg of nuclear protein extracts were used for immunoblot with anti-DDX1 (NovusBiologicals or ProteinTech), anti-AID (Cell Signaling L7E7), anti-Tubulin (Sigma-Aldrich), anti-Actin (Sigma-Aldrich), anti-CTCF (Millipore) or anti-Histone H3 (Abcam) and HRP-conjugated secondary antibodies in TBST, 5% milk (Sigma-Aldrich).

    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: Interactive actions of Bdnf methylation and cell metabolism for building neural resilience under the influence of diet
    Article Snippet: .. Anti-BDNF (1:300; Santa Cruz Biotechnology, CA, USA) and anti-CTCF (1:300, Millipore, MA, USA) antibodies, diluted in 1% bovine serum albumin (BSA)/TBST were added to the cells and incubated overnight at 4°C. .. After thorough washing with PBS, the sections cells were incubated with appropriate dilutions of fluorescent sheep or mouse secondary antibodies (FITC; 1:1000, Cy3; 1:4000; Jackson ImmunoResearch Laboratories Inc., PA, USA) for 1 h at room temperature.

    Infection:

    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: HEK 293T cells were transfected with pSVH for 24 h or BJ-kdLuc and BJ-kdCTCF cells were infected with HCMVdCTCFi, HCMVwt, or HCMVdCTCFiRev at an MOI of 0.5 for 12 h and fixed with 1% formaldehyde and then sonicated to shear the DNA into 100- to 300-bp fragments. .. 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.

    Expressing:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: Co-IP, ChIP and real-time PCR experiments pCDNA3 constructs expressing Flag-tagged CTCF or Flag-tagged RBPJ were transfected into 293T cells. .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    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: CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription
    Article Snippet: .. Western blot analysis Western blot analysis was performed as previously described using approximately 30 µg of proteins harvested from cells boiled in Laemmli buffer (50mM Tris(pH6.8), 2% sodium dodecyl sulfate, 5% β-mercaptoethanol, 10% glycerol, 0.1% bromophenol blue) and antibodies against CTCF (Millipore, 07 729), Nelf-A (Santa cruz, sc-32911), Nelf-E (Santa cruz, sc32912), RNAP II (Santa cruz, sc-9001), Spt5 (Santa cruz, sc-28678), CDK9 (Santa cruz, sc-484), cyclin T1 (Santa cruz, sc-10750), Rad21 (Abcam, ab992), c-Myc (Abcam, 9106), and α-tubulin (Tebu-Bio, 200-301-880). .. Chromatin Immunoprecipitation HeLa cells were transfected using Lipofectamine 2000 (Life Technologies, 11668027) before being subjected to ChIP analysis following the protocol from the Farnham Lab using antibodies against control IgG (Sant cruz, sc-2027), CTCF (Millipore, 07 729), histone H3 (Abcam, ab1791), RNAP II (Santa Cruz, sc-899), NELF-A (Santa Cruz, sc-32911), Spt5 (Santa Cruz, sc-28678), CDK9 (Santa Cruz, sc-8338), cyclin T1 (Santa Cruz, sc-10750), pSer5 (Abcam, 5131) and pSer2 (Abcam, 5095).

    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). ..

    Article Title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination
    Article Snippet: Paragraph title: Protein extracts and western blot analysis ... Typically, 10 μg of whole-cell and 20 μg of nuclear protein extracts were used for immunoblot with anti-DDX1 (NovusBiologicals or ProteinTech), anti-AID (Cell Signaling L7E7), anti-Tubulin (Sigma-Aldrich), anti-Actin (Sigma-Aldrich), anti-CTCF (Millipore) or anti-Histone H3 (Abcam) and HRP-conjugated secondary antibodies in TBST, 5% milk (Sigma-Aldrich).

    Article Title: Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation
    Article Snippet: Paragraph title: Western Blot Analysis. ... 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.

    Article Title: CTCF Regulates Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by Nucleosome Displacement and RNA Polymerase Programming
    Article Snippet: .. The following rabbit polyclonal antibodies were used for the Western blot and ChIP assays: anti-IgG (Santa Cruz Biotechnology), anti-acetylated histone H3 (anti-H3ac) (Millipore), anti-trimethyl histone H3K9, H3K4, H3K36, and H3K79 (Millipore), anti-RNAPII (Abcam), anti-RNAPII S2 (Abcam), anti-RNAPII S5 (Bethyl Laboratories and Abcam), anti-CTCF (Millipore), anti-PTB (Invitrogen), anti-Spt5 (Bethyl Laboratories), anti-proliferating cell nuclear antigen (anti-PCNA) (Abcam), and anti-NELF-A (Bethyl Laboratories). ..

    Article Title: Inhibition of the lytic cycle of Kaposi’s sarcoma-associated herpesvirus by cohesin factors following de novo infection
    Article Snippet: .. The following antibodies were used in ChIPs and/or immunoblots: anti-histone H3 (Abcam ab1791), anti-H3K27me3 (Active Motif #39155), anti-H3K4me3 (Active Motif #39159), anti-EZH2 (Active Motif #39875), anti-RING1B (Abcam ab3832), anti-LANA (Advanced Biotechnologies #13-210-100), anti-CTCF (Millipore, #07-729), anti-RAD21 (Abcam, ab992), anti-SMC3 (Abcam, ab9263), anti-NIPBL (Bethyl Laboratories, A301-779A), anti-K8 (Abcam ab36617), anti-RNA polymerase II (RNAPII) (Abcam), and anti-actin (Abcam). ..

    Transfection:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: Forty-eight hours post transfection cells were lysed in PBS/0.05% Triton X-100/protease inhibitors and sonicated. .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    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: HEK 293T cells were transfected with pSVH for 24 h or BJ-kdLuc and BJ-kdCTCF cells were infected with HCMVdCTCFi, HCMVwt, or HCMVdCTCFiRev at an MOI of 0.5 for 12 h and fixed with 1% formaldehyde and then sonicated to shear the DNA into 100- to 300-bp fragments. .. 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.

    Immunoprecipitation:

    Article Title: Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology
    Article Snippet: For immunoprecipitation, the indicated antibodies were added to pre-cleared lysates along with 40 µl of a 50% slurry of 1∶1 protein A/G beads and incubated at 4°C overnight. .. Antibodies used were: α-CTCF (Millipore), α-Rad21 (Abcam), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz) and α-PAR (Millipore).

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: .. One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs. .. DNA fragments of ∼150 to 300 bp were visualized by agarose gel purification.

    Cell Culture:

    Article Title: Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology
    Article Snippet: Chromatin Immunoprecipitation (ChIP) hESCs, cultured in 10cm dishes, were cross-linked for 15 minutes by addition of formaldehyde (1% final concentration) to TeSR. .. Antibodies used were: α-CTCF (Millipore), α-Rad21 (Abcam), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz) and α-PAR (Millipore).

    Article Title: Interactive actions of Bdnf methylation and cell metabolism for building neural resilience under the influence of diet
    Article Snippet: N2a cells were plated onto 8-chamber slides for immunocytochemical analysis and were cultured for 1 day. .. Anti-BDNF (1:300; Santa Cruz Biotechnology, CA, USA) and anti-CTCF (1:300, Millipore, MA, USA) antibodies, diluted in 1% bovine serum albumin (BSA)/TBST were added to the cells and incubated overnight at 4°C.

    Sequencing:

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs. .. Immunoprecipitated DNA was ligated to adapter primers using the TruSeq ChIP library preparation kit (Illumina) and then sequenced using the Illumina HiSeq 2500 platform according to the manufacturer's recommendations (Illumina) at the Fox Chase Cancer Center Sequencing Facility.

    Sonication:

    Article Title: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: Forty-eight hours post transfection cells were lysed in PBS/0.05% Triton X-100/protease inhibitors and sonicated. .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    Article Title: CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells
    Article Snippet: The sonicated cell supernatant was diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA, and 16.7 mM Tris-HCl, pH 8.1). .. 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.

    Article Title: Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology
    Article Snippet: Lysates were subsequently sonicated and 500 µg of lysate was pre-cleared for 1 hour using 40 µl of a 50% slurry of 1∶1 protein A- and protein G-Sepharose beads (GE Healthcare). .. Antibodies used were: α-CTCF (Millipore), α-Rad21 (Abcam), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz) and α-PAR (Millipore).

    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: HEK 293T cells were transfected with pSVH for 24 h or BJ-kdLuc and BJ-kdCTCF cells were infected with HCMVdCTCFi, HCMVwt, or HCMVdCTCFiRev at an MOI of 0.5 for 12 h and fixed with 1% formaldehyde and then sonicated to shear the DNA into 100- to 300-bp fragments. .. 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: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: For XChIP on crosslinked preparations, formaldehyde-fixed NeuN+ nuclei after FACS were resuspended in lysis buffer containing 0.1%SDS, sonicated ( Bioruptor® Plus sonication device , Diagenode) at the ‘high’ setting for 30 minutes on ice. .. Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads.

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: .. One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs. .. DNA fragments of ∼150 to 300 bp were visualized by agarose gel purification.

    Binding Assay:

    Article Title: Interactive actions of Bdnf methylation and cell metabolism for building neural resilience under the influence of diet
    Article Snippet: After 24 h of treatment, cells were fixed with 4% para-formaldehyde for 10 min, washed with PBS and incubated for 1 h at room temperature with TBS solution containing 3% BSA and 0.3% Triton X-100 to inhibit non-specific binding. .. Anti-BDNF (1:300; Santa Cruz Biotechnology, CA, USA) and anti-CTCF (1:300, Millipore, MA, USA) antibodies, diluted in 1% bovine serum albumin (BSA)/TBST were added to the cells and incubated overnight at 4°C.

    Immunofluorescence:

    Article Title: Characterization of DXZ4 conservation in primates implies important functional roles for CTCF binding, array expression and tandem repeat organization on the X chromosome
    Article Snippet: .. Immunofluorescence on interphase cells and metaphase chromosomes Rabbit polyclonal anti-H3K4me2 (07-030), anti-H3K4me3 (05-745), anti-CTCF (07-729), anti-H3K36me2 (07-274) and anti-acetyl-lysine (06-933) were all obtained from Millipore (Billerica, MA, USA). .. Mouse monoclonal anti-H3K27me3 (ab6002) was obtained from Abcam (Cambridge, MA, USA).

    ChIP-sequencing:

    Article Title: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: Paragraph title: ChIP-seq ... Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads.

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: Paragraph title: ChIP-seq. ... One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs.

    Magnetic Beads:

    Article Title: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: .. Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads. .. After washing and elution, DNA was incubated at 65°C overnight for reverse cross-linking, followed by RNase A, proteinase K treatment and DNA precipitation.

    Isolation:

    Article Title: CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells
    Article Snippet: Briefly, cross-linked cells (1% formaldehyde) were isolated and lysed in lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl, pH 8.1) containing protease inhibitors, followed by sonication on ice until the cross-linked chromatin DNA was sheared to a size range of 200 to 1,000 bp. .. 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.

    Clarification 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: After clarification of the soluble fraction by centrifugation, lysates were incubated overnight with anti-Flag (M2) agarose (Sigma) at 4°C. .. Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    Purification:

    Article Title: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: DNA was purified by phenol-chloroform extraction and ethanol precipitation. .. Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads.

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs. .. DNA fragments of ∼150 to 300 bp were visualized by agarose gel purification.

    Polymerase Chain Reaction:

    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 PCR products were quantified with normalization to the input DNA.

    FACS:

    Article Title: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: For XChIP on crosslinked preparations, formaldehyde-fixed NeuN+ nuclei after FACS were resuspended in lysis buffer containing 0.1%SDS, sonicated ( Bioruptor® Plus sonication device , Diagenode) at the ‘high’ setting for 30 minutes on ice. .. Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads.

    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: RBPJ, the Major Transcriptional Effector of Notch Signaling, Remains Associated with Chromatin throughout Mitosis, Suggesting a Role in Mitotic Bookmarking
    Article Snippet: Paragraph title: Co-IP, ChIP and real-time PCR experiments ... Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

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

    Article Title: Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) ... Antibodies used were: α-CTCF (Millipore), α-Rad21 (Abcam), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz) and α-PAR (Millipore).

    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: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads. .. Setdb1 occupancies were measured by conventional ChIP-PCR.

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: Chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) was performed as previously described ( , ), with minor changes. .. One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs.

    Article Title: Higher-Order Chromatin Regulation and Differential Gene Expression in the Human Tumor Necrosis Factor/Lymphotoxin Locus in Hepatocellular Carcinoma Cells
    Article Snippet: .. In order to clarify the localization of CTCF and the cofactor cohesin RAD21 in hepatic cells, we performed chromatin immunoprecipitation (ChIP) analyses using anti-CTCF and anti-RAD21 antibodies, followed by quantitative PCR (qPCR) ( C and D). ..

    Article Title: CTCF Regulates Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by Nucleosome Displacement and RNA Polymerase Programming
    Article Snippet: .. The following rabbit polyclonal antibodies were used for the Western blot and ChIP assays: anti-IgG (Santa Cruz Biotechnology), anti-acetylated histone H3 (anti-H3ac) (Millipore), anti-trimethyl histone H3K9, H3K4, H3K36, and H3K79 (Millipore), anti-RNAPII (Abcam), anti-RNAPII S2 (Abcam), anti-RNAPII S5 (Bethyl Laboratories and Abcam), anti-CTCF (Millipore), anti-PTB (Invitrogen), anti-Spt5 (Bethyl Laboratories), anti-proliferating cell nuclear antigen (anti-PCNA) (Abcam), and anti-NELF-A (Bethyl Laboratories). ..

    SDS Page:

    Article Title: Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation
    Article Snippet: Proteins were resolved by 4–12% (wt/vol) SDS/PAGE (Invitrogen), transferred to PVDF membranes (Osmonics; GE), and blocked with 5% (wt/vol) skim milk powder in 0.1% (vol/vol) Tween 20-PBS for 1 h at room temperature. .. 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.

    SYBR Green Assay:

    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. .. All samples were analyzed in triplicate with SYBR green 2× master mix (Applied Biosystems, Foster City, CA) on an Applied Biosystems 7900HT Fast real-time PCR system.

    Co-Immunoprecipitation 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: Paragraph title: Co-IP, ChIP and real-time PCR experiments ... Western blots were probed with a rabbit anti-CTCF antibody (Millipore Cat#07-729) or the rabbit anti-RBPJ antibody.

    Agarose Gel Electrophoresis:

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs. .. DNA fragments of ∼150 to 300 bp were visualized by agarose gel purification.

    Ethanol Precipitation:

    Article Title: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: DNA was purified by phenol-chloroform extraction and ethanol precipitation. .. Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads.

    Next-Generation Sequencing:

    Article Title: PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type
    Article Snippet: Chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) was performed as previously described ( , ), with minor changes. .. One-tenth of the sonicated chromatin was collected and used as input material, while the rest of the chromatin was immunoprecipitated using 5 μg of CTCF antibody (Millipore), 5 μg of PARP1 antibody (Active Motif), or 5 μg of normal rabbit IgG (Santa Cruz) in LCLs.

    Concentration Assay:

    Article Title: Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology
    Article Snippet: Cross-linking was stopped upon addition of Glycine to a final concentration of 125 mM for 5 minutes. .. Antibodies used were: α-CTCF (Millipore), α-Rad21 (Abcam), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz) and α-PAR (Millipore).

    Lysis:

    Article Title: CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells
    Article Snippet: Briefly, cross-linked cells (1% formaldehyde) were isolated and lysed in lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl, pH 8.1) containing protease inhibitors, followed by sonication on ice until the cross-linked chromatin DNA was sheared to a size range of 200 to 1,000 bp. .. 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.

    Article Title: Kmt1e regulates a large neuron-specific topological chromatin domain
    Article Snippet: For XChIP on crosslinked preparations, formaldehyde-fixed NeuN+ nuclei after FACS were resuspended in lysis buffer containing 0.1%SDS, sonicated ( Bioruptor® Plus sonication device , Diagenode) at the ‘high’ setting for 30 minutes on ice. .. Chromatin was then incubated with anti-CTCF (EMD Millipore, # 07729) or anti-SETDB1 (Santa Cruz H-300X ##sc-66884 X) or anti-SETDB1 (Thermofisher 5H6A12 #MA5-15722) and captured with Protein AG Magnetic Beads.

    Article Title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination
    Article Snippet: For nuclear protein extracts, 10x106 cells were resuspended in 500 μL cell lysis buffer (50 mM Tris-HCl, pH 8, 300 mM Sucrose, 4 mM MgAc, 12.5 mM KCl, 1 mM DTT, 10 mM β-mercaptoethanol, 0.3% NP-40 and 1mM PMSF) and incubated 5 min on ice followed by centrifugation (500 g for 10 min at 4°C). .. Typically, 10 μg of whole-cell and 20 μg of nuclear protein extracts were used for immunoblot with anti-DDX1 (NovusBiologicals or ProteinTech), anti-AID (Cell Signaling L7E7), anti-Tubulin (Sigma-Aldrich), anti-Actin (Sigma-Aldrich), anti-CTCF (Millipore) or anti-Histone H3 (Abcam) and HRP-conjugated secondary antibodies in TBST, 5% milk (Sigma-Aldrich).

    Staining:

    Article Title: Characterization of DXZ4 conservation in primates implies important functional roles for CTCF binding, array expression and tandem repeat organization on the X chromosome
    Article Snippet: Immunofluorescence on interphase cells and metaphase chromosomes Rabbit polyclonal anti-H3K4me2 (07-030), anti-H3K4me3 (05-745), anti-CTCF (07-729), anti-H3K36me2 (07-274) and anti-acetyl-lysine (06-933) were all obtained from Millipore (Billerica, MA, USA). .. Cell staining and preparation of metaphase chromosomes was performed essentially as described [ ].

    Article Title: Interactive actions of Bdnf methylation and cell metabolism for building neural resilience under the influence of diet
    Article Snippet: Anti-BDNF (1:300; Santa Cruz Biotechnology, CA, USA) and anti-CTCF (1:300, Millipore, MA, USA) antibodies, diluted in 1% bovine serum albumin (BSA)/TBST were added to the cells and incubated overnight at 4°C. .. The results of immunocytochemistry controls were negative as no staining was observed in cell structures.

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  • 90
    Millipore rabbit anti ctcf
    <t>CSB</t> regulates a subset of <t>CTCF</t> 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
    Rabbit Anti Ctcf, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti ctcf/product/Millipore
    Average 90 stars, based on 3 article reviews
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    90
    Millipore ctcf
    Validation of DNA binding protein occupancy. ChIP analysis of K562 chromatin for occupancy by the erythroid-specific transcription factors GATA1 and NF-E2; <t>RNA</t> Pol II; the chromatin architectural protein <t>CTCF;</t> and the barrier-associated proteins USF1,
    Ctcf, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ctcf - by Bioz Stars, 2020-02
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    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

    Validation of DNA binding protein occupancy. ChIP analysis of K562 chromatin for occupancy by the erythroid-specific transcription factors GATA1 and NF-E2; RNA Pol II; the chromatin architectural protein CTCF; and the barrier-associated proteins USF1,

    Journal: Blood

    Article Title: A tissue-specific chromatin loop activates the erythroid ankyrin-1 promoter

    doi: 10.1182/blood-2012-08-450262

    Figure Lengend Snippet: Validation of DNA binding protein occupancy. ChIP analysis of K562 chromatin for occupancy by the erythroid-specific transcription factors GATA1 and NF-E2; RNA Pol II; the chromatin architectural protein CTCF; and the barrier-associated proteins USF1,

    Article Snippet: ChIP was performed using the Magna ChIP A assay kit (17-610; Millipore), with anti-GATA1 (sc-265), –NF-E2 (sc-22 827), -USF2 (sc-862), -USF1 (sc-8983), -PRMT1, and -RNA polymerase II (sc-130851; all from Santa Cruz Biotechnology), -CTCF (07-729; Millipore), or -CARM1 (PRMT4, ab84370; Abcam) antibodies.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    Knocking down of CTCF results in increased cell-to-cell variation FACS analysis revealed increased cell-to-cell variation of expression of GATA3 (A), CD28 (B), CD90 (C), and CD5 (D). No significantly changed variation of expression was detected for Cohesin (E). Left panels show the distribution of gene expression with the x-axis indicating the expression level and y-axis indicating the cell density. Right panels are bar plots for the coefficient of variation that measures the expression variation of cells from individual replicate. APC signal were log10 transformed. Replicates for KD cells and control cells were paired based on experiment date. P-value was obtained by paired t-test.

    Journal: Molecular cell

    Article Title: CTCF-mediated enhancer-promoter interaction is a critical regulator of cell-to-cell variation of gene expression

    doi: 10.1016/j.molcel.2017.08.026

    Figure Lengend Snippet: Knocking down of CTCF results in increased cell-to-cell variation FACS analysis revealed increased cell-to-cell variation of expression of GATA3 (A), CD28 (B), CD90 (C), and CD5 (D). No significantly changed variation of expression was detected for Cohesin (E). Left panels show the distribution of gene expression with the x-axis indicating the expression level and y-axis indicating the cell density. Right panels are bar plots for the coefficient of variation that measures the expression variation of cells from individual replicate. APC signal were log10 transformed. Replicates for KD cells and control cells were paired based on experiment date. P-value was obtained by paired t-test.

    Article Snippet: The following antibodies were used for ChIP-Seq analysis: H3K27ac (ab4729, Abcam); Cohesin (SMC1) (A300-055A, Bethyl), CTCF (07-729, Millipore), RNA Polymerase II (ab5408, Abcam).

    Techniques: FACS, Expressing, Transformation Assay

    CTCF binding sites interact with enhancers and promoters and positively correlate with gene activation A. Interaction density among regulatory elements positively correlates with CTCF, Cohesin, GATA3, p300 and active histone modifications. The high-confidence interacting regions were sorted based on their interaction density (red: high; blue: low). The binding levels of chromatin proteins and histone modifications determined by ChIP-Seq were plotted as indicated at the bottom of the panel. B. Scatter plot shows a positive correlation between interaction density and H3K27ac level. C. Scatter plot shows a positive correlation between interaction density and CTCF binding level. D. Scatter plot shows a positive correlation between levels of CTCF binding and H3K27ac modification. E. More active genes are bound by CTCF than silent genes. The fraction of active or inactive promoters (+/− 2kb around TSS) bound by CTCF is plotted. F. Distribution of the closest p300 site relative to the CTCF sites in the genome. The location of CTCF sites is indicated by the arrow at bottom. The closest p300 sites are found and p300 binding levels are plotted (red: high; green: low). Displayed are the p300 sites located from 3 to 20kb away from the CTCF sites. G. Interaction density peaks at the CTCF and p300 binding sites. The interaction densities for the chromatin regions described in Panel F above are plotted. H. p300 sites interact with their neighboring CTCF sites. Plotted are the fractions of p300 sites that interact with the nearest CTCF sites. The background shows the interaction density with the chromatin regions without p300 binding at equal distance. I. Active promoters exhibit more looping with distal CTCF sites than silent promoters. Genes were separated to active genes (RPKM ≥3) and silent genes (RPKM ≤3). The number of CTCF binding sites ( > 5kb from TSS), which interact with the promoters, is plotted per promoter on Y-axis. J. Enhancers near or interacting with CTCF sites are more interactive. The p300-bound enhancers are separated to three categories: (1) with at least one CTCF site within a distance of 20 kb; (2) interacting with a CTCF site; and (3) others. The number of interacting promoters or enhancers is plotted for each category (Y-axis).

    Journal: Molecular cell

    Article Title: CTCF-mediated enhancer-promoter interaction is a critical regulator of cell-to-cell variation of gene expression

    doi: 10.1016/j.molcel.2017.08.026

    Figure Lengend Snippet: CTCF binding sites interact with enhancers and promoters and positively correlate with gene activation A. Interaction density among regulatory elements positively correlates with CTCF, Cohesin, GATA3, p300 and active histone modifications. The high-confidence interacting regions were sorted based on their interaction density (red: high; blue: low). The binding levels of chromatin proteins and histone modifications determined by ChIP-Seq were plotted as indicated at the bottom of the panel. B. Scatter plot shows a positive correlation between interaction density and H3K27ac level. C. Scatter plot shows a positive correlation between interaction density and CTCF binding level. D. Scatter plot shows a positive correlation between levels of CTCF binding and H3K27ac modification. E. More active genes are bound by CTCF than silent genes. The fraction of active or inactive promoters (+/− 2kb around TSS) bound by CTCF is plotted. F. Distribution of the closest p300 site relative to the CTCF sites in the genome. The location of CTCF sites is indicated by the arrow at bottom. The closest p300 sites are found and p300 binding levels are plotted (red: high; green: low). Displayed are the p300 sites located from 3 to 20kb away from the CTCF sites. G. Interaction density peaks at the CTCF and p300 binding sites. The interaction densities for the chromatin regions described in Panel F above are plotted. H. p300 sites interact with their neighboring CTCF sites. Plotted are the fractions of p300 sites that interact with the nearest CTCF sites. The background shows the interaction density with the chromatin regions without p300 binding at equal distance. I. Active promoters exhibit more looping with distal CTCF sites than silent promoters. Genes were separated to active genes (RPKM ≥3) and silent genes (RPKM ≤3). The number of CTCF binding sites ( > 5kb from TSS), which interact with the promoters, is plotted per promoter on Y-axis. J. Enhancers near or interacting with CTCF sites are more interactive. The p300-bound enhancers are separated to three categories: (1) with at least one CTCF site within a distance of 20 kb; (2) interacting with a CTCF site; and (3) others. The number of interacting promoters or enhancers is plotted for each category (Y-axis).

    Article Snippet: The following antibodies were used for ChIP-Seq analysis: H3K27ac (ab4729, Abcam); Cohesin (SMC1) (A300-055A, Bethyl), CTCF (07-729, Millipore), RNA Polymerase II (ab5408, Abcam).

    Techniques: Binding Assay, Activation Assay, Chromatin Immunoprecipitation, Modification

    CTCF binding sites at the Thy1 locus contribute to the functional interaction between Thy1 promoter and its enhancers and the expression noise control. CD90 protein is encoded by the Thy1 gene A. The chromatin interactions surrounding the Thy1 gene locus are shown in the upper panel and H3K4me2, H3K4me3, H3K27ac and CTCF ChIP-Seq signals are shown in the lower panels. The red rectangle highlights the interactions between the CTCF binding site and Thy1 promoter and enhancers. The CTCF binding sites high-lighted by scissors were deleted separately in EL4 cells using CRISPR/CAS9. B. Deletion of the 1 st CTCF binding site decreased Thy1 mRNA levels. Total RNAs isolated from the wild type or CTCF site deletion EL4 clones were analyzed by quantitative reverse-transcription PCR and normalized to GAPDH. C. The 1 st CTCF site deletion abolished CTCF binding and compromised the H3K27ac modification at the Thy1 gene locus. The genome browser images show the ChIP-Seq data for CTCF binding, H3K27ac and chromatin input signals in wild type and CRISPR deletion EL4 cells. The high-lighted CTCF peak indicates the location of CRISPR deletion. D. The 1 st CTCF site deletion compromised the enhancer-promoter interaction in the Thy1 gene locus. Top panel shows the H3K4me2 and H3K27ac ChIP-Seq signals. The red and green horizontal lines below the ChIP-Seq tracks indicate the different TADs called by HMM. The bottom panel shows the relative chromatin interaction intensity of the Thy1 promoter with various enhancer regions indicated above the top panel (R1 to R10) by 3C analysis. The blue rectangle marked as R6 is the anchor site for the 3C analysis. The red rectangle marked R8 region is the deleted 1 st CTCF site. Data show average of two independent experiments and are represented as mean ± SEM. WT: control cells; KO: CRISPR/CAS9 deletion cells. E. Deletion of the 1 st CTCF site resulted in increased cell-to-cell variation of expression of CD90 protein encoded by the Thy1 gene as measured by FACS assay.

    Journal: Molecular cell

    Article Title: CTCF-mediated enhancer-promoter interaction is a critical regulator of cell-to-cell variation of gene expression

    doi: 10.1016/j.molcel.2017.08.026

    Figure Lengend Snippet: CTCF binding sites at the Thy1 locus contribute to the functional interaction between Thy1 promoter and its enhancers and the expression noise control. CD90 protein is encoded by the Thy1 gene A. The chromatin interactions surrounding the Thy1 gene locus are shown in the upper panel and H3K4me2, H3K4me3, H3K27ac and CTCF ChIP-Seq signals are shown in the lower panels. The red rectangle highlights the interactions between the CTCF binding site and Thy1 promoter and enhancers. The CTCF binding sites high-lighted by scissors were deleted separately in EL4 cells using CRISPR/CAS9. B. Deletion of the 1 st CTCF binding site decreased Thy1 mRNA levels. Total RNAs isolated from the wild type or CTCF site deletion EL4 clones were analyzed by quantitative reverse-transcription PCR and normalized to GAPDH. C. The 1 st CTCF site deletion abolished CTCF binding and compromised the H3K27ac modification at the Thy1 gene locus. The genome browser images show the ChIP-Seq data for CTCF binding, H3K27ac and chromatin input signals in wild type and CRISPR deletion EL4 cells. The high-lighted CTCF peak indicates the location of CRISPR deletion. D. The 1 st CTCF site deletion compromised the enhancer-promoter interaction in the Thy1 gene locus. Top panel shows the H3K4me2 and H3K27ac ChIP-Seq signals. The red and green horizontal lines below the ChIP-Seq tracks indicate the different TADs called by HMM. The bottom panel shows the relative chromatin interaction intensity of the Thy1 promoter with various enhancer regions indicated above the top panel (R1 to R10) by 3C analysis. The blue rectangle marked as R6 is the anchor site for the 3C analysis. The red rectangle marked R8 region is the deleted 1 st CTCF site. Data show average of two independent experiments and are represented as mean ± SEM. WT: control cells; KO: CRISPR/CAS9 deletion cells. E. Deletion of the 1 st CTCF site resulted in increased cell-to-cell variation of expression of CD90 protein encoded by the Thy1 gene as measured by FACS assay.

    Article Snippet: The following antibodies were used for ChIP-Seq analysis: H3K27ac (ab4729, Abcam); Cohesin (SMC1) (A300-055A, Bethyl), CTCF (07-729, Millipore), RNA Polymerase II (ab5408, Abcam).

    Techniques: Binding Assay, Functional Assay, Expressing, Chromatin Immunoprecipitation, CRISPR, Isolation, Clone Assay, Polymerase Chain Reaction, Modification, FACS

    Single-molecule RNA-FISH shows increased cell-to-cell variation of Thy1 mRNA in the CTCF site-deleted cells A. Typical images of RNA-FISH for detecting CTCF mRNA (red), Thy1 mRNA (green) and DNA (blue). B. Box plots showing the numbers of CTCF and Thy1 mRNA molecules per cell in wild type EL4 cells. The box plot is from one representative of 4 replicates. C. Box plots showing the numbers of CTCF and Thy1 mRNA molecules per cell in the 1 st CTCF site-deleted EL4 cells. The box plot is from one representative of 12 replicates. D. Deletion of the 1 st CTCF site results in increased coefficient of variation in the number of Thy1 mRNAs per cell. The bar plot shows the distribution of CVs of 4 replicates for WT and 12 replicates for KO. Data represented as mean ± SEM. P-value was obtained by t-test. E. The variation of Thy1 mRNA per cell caused by deletion of the 1 st CTCF site is related to the number of CTCF mRNA in the cell. On the X-axis, the cells are sorted to four groups according to the number of CTCF mRNAs per cell (0–14; 15–29; 30–44; and > 44). Y-axis indicates the coefficient of variation of Thy1 mRNA. Grey bars indicate the wild type EL4 cells and red bars indicate the CTCF site deletion EL4 cells. Each bar represents one replicate. F. CTCF and Cohesin organize chromatin to large domains (left panel) and facilitate long-distance enhancer-promoter interaction to decrease fluctuation of expression and maintain robustness of expression (right panel).

    Journal: Molecular cell

    Article Title: CTCF-mediated enhancer-promoter interaction is a critical regulator of cell-to-cell variation of gene expression

    doi: 10.1016/j.molcel.2017.08.026

    Figure Lengend Snippet: Single-molecule RNA-FISH shows increased cell-to-cell variation of Thy1 mRNA in the CTCF site-deleted cells A. Typical images of RNA-FISH for detecting CTCF mRNA (red), Thy1 mRNA (green) and DNA (blue). B. Box plots showing the numbers of CTCF and Thy1 mRNA molecules per cell in wild type EL4 cells. The box plot is from one representative of 4 replicates. C. Box plots showing the numbers of CTCF and Thy1 mRNA molecules per cell in the 1 st CTCF site-deleted EL4 cells. The box plot is from one representative of 12 replicates. D. Deletion of the 1 st CTCF site results in increased coefficient of variation in the number of Thy1 mRNAs per cell. The bar plot shows the distribution of CVs of 4 replicates for WT and 12 replicates for KO. Data represented as mean ± SEM. P-value was obtained by t-test. E. The variation of Thy1 mRNA per cell caused by deletion of the 1 st CTCF site is related to the number of CTCF mRNA in the cell. On the X-axis, the cells are sorted to four groups according to the number of CTCF mRNAs per cell (0–14; 15–29; 30–44; and > 44). Y-axis indicates the coefficient of variation of Thy1 mRNA. Grey bars indicate the wild type EL4 cells and red bars indicate the CTCF site deletion EL4 cells. Each bar represents one replicate. F. CTCF and Cohesin organize chromatin to large domains (left panel) and facilitate long-distance enhancer-promoter interaction to decrease fluctuation of expression and maintain robustness of expression (right panel).

    Article Snippet: The following antibodies were used for ChIP-Seq analysis: H3K27ac (ab4729, Abcam); Cohesin (SMC1) (A300-055A, Bethyl), CTCF (07-729, Millipore), RNA Polymerase II (ab5408, Abcam).

    Techniques: Fluorescence In Situ Hybridization, Expressing

    RARRES1 expression is influenced by DNA methylation and retinoic acid signaling A. The effect of decitabine (DAC) and retinoic acid (RA), alone or combined on RARRES1 expression was determined by qPCR in methylated cell lines (MDA-MB-231, MDA-MB-436, HCC1599), and unmethylated MDA-MB-468 cells. Treatments were compared using a repeated-measures ANOVA. B. The effect of ALDH1A3 overexpression in MDA-MB-231 cells (have low levels of intrinsic ALDH1A3) and ALDH1A3 knockdown in MDA-MB-468 cells (have high levels of intrinsic ALDH1A3) on RARRES1 expression was determined by qPCR. Decitabine-treated values were compared to no-treatment values using a paired student's t-test. C. To interrogate the RARRES1 promoter, ChIP and double ChIP assays on were performed on MDA-MB-231 cells (have methylated RARRES1 promoter and low levels of ALDH1A3, which produces RA) and MDA-MB-468 cells (have unmethylated RARRES1 promoter and intrinsic high ALDH1A3) that were either treated with decitabine (DAC), retinoic acid (RA) or both. The assays were performed using antibodies against 5-mC, RARα and CTCF, as well as the control normal rabbit IgG, alone or in combination for the double ChIP assays. In the double ChIP assays only DNA sequences that bind both proteins concurrently are detected by this assay.

    Journal: Oncotarget

    Article Title: Breast cancer subtype dictates DNA methylation and ALDH1A3-mediated expression of tumor suppressor RARRES1

    doi: 10.18632/oncotarget.9858

    Figure Lengend Snippet: RARRES1 expression is influenced by DNA methylation and retinoic acid signaling A. The effect of decitabine (DAC) and retinoic acid (RA), alone or combined on RARRES1 expression was determined by qPCR in methylated cell lines (MDA-MB-231, MDA-MB-436, HCC1599), and unmethylated MDA-MB-468 cells. Treatments were compared using a repeated-measures ANOVA. B. The effect of ALDH1A3 overexpression in MDA-MB-231 cells (have low levels of intrinsic ALDH1A3) and ALDH1A3 knockdown in MDA-MB-468 cells (have high levels of intrinsic ALDH1A3) on RARRES1 expression was determined by qPCR. Decitabine-treated values were compared to no-treatment values using a paired student's t-test. C. To interrogate the RARRES1 promoter, ChIP and double ChIP assays on were performed on MDA-MB-231 cells (have methylated RARRES1 promoter and low levels of ALDH1A3, which produces RA) and MDA-MB-468 cells (have unmethylated RARRES1 promoter and intrinsic high ALDH1A3) that were either treated with decitabine (DAC), retinoic acid (RA) or both. The assays were performed using antibodies against 5-mC, RARα and CTCF, as well as the control normal rabbit IgG, alone or in combination for the double ChIP assays. In the double ChIP assays only DNA sequences that bind both proteins concurrently are detected by this assay.

    Article Snippet: Chromatin immunoprecipitation ChIP assays [ ] were performed following the ChIP assay kit protocol (cat#06-599, Upstate Biotechnology) as previously described [ ] using antibodies against 5-mC (cat#BI-MECY-0500, AnaSpec, Inc.), RARα (cat#ab41934, abcam), CTCF (cat#07-729, Millipore) as well as the control normal rabbit IgG (cat#sc-2027, Santa Cruz Biotechnology).

    Techniques: Expressing, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Methylation, Multiple Displacement Amplification, Over Expression, Chromatin Immunoprecipitation