anti ctcf rabbit monoclonal antibody  (Cell Signaling Technology Inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Name:
    CTCF D1A7 XP Rabbit mAb
    Description:
    CCCTC binding factor CTCF and its paralog the Brother of the Regulator of Imprinted Sites BORIS are highly conserved transcription factors that regulate transcriptional activation and repression insulator function and imprinting control regions ICRs 1 4 Although they have divergent amino and carboxy termini both proteins contain 11 conserved zinc finger domains that work in combination to bind the same DNA elements 1 CTCF is ubiquitously expressed and contributes to transcriptional regulation of cell growth regulated genes including c myc p19 ARF p16 INK4A BRCA1 p53 p27 E2F1 and TERT 1 CTCF also binds to and is required for the enhancer blocking activity of all known insulator elements and ICRs including the H19 IgF2 Prader Willi Angelman syndrome and Inactive X Specific Transcript XIST anti sense loci 5 7 CTCF DNA binding is sensitive to DNA methylation a mark that determines selection of the imprinted allele maternal vs paternal 1 The various functions of CTCF are regulated by at least two different post translational modifications Poly ADP ribosyl ation of CTCF is required for insulator function 8 Phosphorylation of Ser612 by protein kinase CK2 facilitates a switch of CTCF from a transcriptional repressor to an activator at the c myc promoter 9 CTCF mutations or deletions have been found in many breast prostate and Wilms tumors 10 11 Expression of BORIS is restricted to spermatocytes and is mutually exclusive of CTCF 3 In cells expressing BORIS promoters of X linked cancer testis antigens like MAGE 1A are demethylated and activated but methylated and inactive in CTCF expressing somatic cells 12 Like other testis specific proteins BORIS is abnormally expressed in different cancers such as breast cancer and has a greater affinity than CTCF for DNA binding sites detracting from CTCF s potential tumor suppressing activity 1 3 13 14
    Catalog Number:
    3417
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the human CTCF protein.
    Reactivity:
    Human Rat Monkey
    Applications:
    Western Blot, Immunoprecipitation, Immunofluorescence, Chromatin Immunoprecipitation
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti ctcf rabbit monoclonal antibody
    Transcriptional profiling of genes altered with <t>CTCF</t> knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF <t>shRNA</t> induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR
    CCCTC binding factor CTCF and its paralog the Brother of the Regulator of Imprinted Sites BORIS are highly conserved transcription factors that regulate transcriptional activation and repression insulator function and imprinting control regions ICRs 1 4 Although they have divergent amino and carboxy termini both proteins contain 11 conserved zinc finger domains that work in combination to bind the same DNA elements 1 CTCF is ubiquitously expressed and contributes to transcriptional regulation of cell growth regulated genes including c myc p19 ARF p16 INK4A BRCA1 p53 p27 E2F1 and TERT 1 CTCF also binds to and is required for the enhancer blocking activity of all known insulator elements and ICRs including the H19 IgF2 Prader Willi Angelman syndrome and Inactive X Specific Transcript XIST anti sense loci 5 7 CTCF DNA binding is sensitive to DNA methylation a mark that determines selection of the imprinted allele maternal vs paternal 1 The various functions of CTCF are regulated by at least two different post translational modifications Poly ADP ribosyl ation of CTCF is required for insulator function 8 Phosphorylation of Ser612 by protein kinase CK2 facilitates a switch of CTCF from a transcriptional repressor to an activator at the c myc promoter 9 CTCF mutations or deletions have been found in many breast prostate and Wilms tumors 10 11 Expression of BORIS is restricted to spermatocytes and is mutually exclusive of CTCF 3 In cells expressing BORIS promoters of X linked cancer testis antigens like MAGE 1A are demethylated and activated but methylated and inactive in CTCF expressing somatic cells 12 Like other testis specific proteins BORIS is abnormally expressed in different cancers such as breast cancer and has a greater affinity than CTCF for DNA binding sites detracting from CTCF s potential tumor suppressing activity 1 3 13 14
    https://www.bioz.com/result/anti ctcf rabbit monoclonal antibody/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ctcf rabbit monoclonal antibody - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "CTCF loss mediates unique DNA hypermethylation landscapes in human cancers"

    Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-020-00869-7

    Transcriptional profiling of genes altered with CTCF knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF shRNA induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR
    Figure Legend Snippet: Transcriptional profiling of genes altered with CTCF knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF shRNA induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR

    Techniques Used: shRNA

    Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P
    Figure Legend Snippet: Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P

    Techniques Used: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation, shRNA, Western Blot

    Related Articles

    Western Blot:

    Article Title: BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells
    Article Snippet: .. The elution step was conducted by heating the beads for 10 min at 95 °C in lithium dodecyl sulfate (LDS) sample buffer with reducing agent (Invitrogen), after which western blotting was performed using the following antibodies: CTCF (3417, Cell Signaling Technology) and BORIS (9851, Active Motif). ..

    Article Title: CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair
    Article Snippet: .. The antibodies used were CTCF (rabbit, Cell Signaling, 3417 S, 1:100), SUZ12 (rabbit, Cell Signaling Technology, 3737 S, 1:1000 for Western), anti-Flag (rabbit, 14793 S or mouse, 8146 S, Cell Signaling Technology, 1:200), and HA-tag (mouse, Cell Signaling, 2367 S, 1:200). ..

    Immunoprecipitation:

    Article Title: BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells
    Article Snippet: .. Antibodies used for immunoprecipitation were CTCF (3417, Cell Signaling Technology) and BORIS (NBP2–52405, NOVUS Biologicals). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Cell Signaling Technology Inc anti ctcf rabbit monoclonal antibody
    Transcriptional profiling of genes altered with <t>CTCF</t> knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF <t>shRNA</t> induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR
    Anti Ctcf Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ctcf rabbit monoclonal antibody/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ctcf rabbit monoclonal antibody - by Bioz Stars, 2021-09
    93/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc ctcf
    HLA Class II chromatin landscapes of HLA-DR3 and DR15 haplotypes. a <t>CTCF</t> HiChIP looping interactions within the HLA Class II region reveal an insulated neighborhood that extends from BTNL2 to upstream of HLA-DQA2 . The COX-aligned DR3 haplotype is presented on top and the PGF-aligned DR15 haplotype is presented on the bottom. Gene and bp positions are presented specific for each haplotype. Orange arcs represent CTCF-mediated HiChIP interactions. Arc thickness is proportional to the frequency of observed paired-end tags (PETs) at each enhancer. b <t>H3K27ac</t> HiChIP looping interactions within the HLA Class II region reveal differential frequency and pattern in the HLA-DRB1 to HLA-DQB1 region between the HLA-DR15 and HLA-DR3 haplotypes. Black arcs represent H3K27ac-mediated HiChIP interactions. Significant p -values are presented for enhancers that exhibit differential binding frequencies (mean PETs) for one haplotype or the other and are colored by the haplotype that exhibits higher PET counts (HLA-DR3: blue; HLA-DR15: red). H3K27ac ChIP peaks are presented in blue (HLA-DR3) and red (HLA-DR15). Haplotype specific loops are indicated in blue (COX) or red (PGF). Corresponding haplotype specific enhancers are labeled as follows—COX: 1: 4.007–4.013 Mb, 2: 4.021–4.031 Mb, 3: 4.039–4.047 Mb, 4: 4.052–4.06 Mb, 5: 4.078–4.083 Mb; PGF: 1: 4.076–4.081 Mb, B : 4.092–4.102 Mb, C : 4.111–4.120 Mb, D : 4.124–4.129 Mb, E : 4.154–4.159 Mb. c Individual H3K27 HiChIP looping interactions for three subjects heterozygous for HLA-DR3 (left) and three subjects homozygous for HLA-DR15 (right). H3K27ac ChIP peaks, haplotype specific loops, and haplotype specific enhancers are presented in blue (HLA-DR3) and red (HLA-DR15)
    Ctcf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ctcf/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ctcf - by Bioz Stars, 2021-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Transcriptional profiling of genes altered with CTCF knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF shRNA induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR

    Journal: Clinical Epigenetics

    Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

    doi: 10.1186/s13148-020-00869-7

    Figure Lengend Snippet: Transcriptional profiling of genes altered with CTCF knockdown. a Heat map of differentially expressed (DE) transcripts following 5 days of CTCF shRNA induction (Dox) versus uninduced vehicle control (vehicle). CTCF knockdown in HPECE6/E7 leads to 1308 significantly altered gene transcripts (FDR

    Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

    Techniques: shRNA

    Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P

    Journal: Clinical Epigenetics

    Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

    doi: 10.1186/s13148-020-00869-7

    Figure Lengend Snippet: Knockdown of CTCF protein results in DNA hypermethylation preferentially at CTCF sites. a Workflow of methylated DNA immunoprecipitation followed by copy number array application (MeDIP-chip) for detecting methylation alterations. NspI restriction fragments were bound to anti-5-methylcytosine antibody, eluted, and hybridized to a Affymetrix Cytoscan HD probe. An unenriched total input fraction was processed for comparison. b Short hairpin mediated CTCF knockdown in two separate shRNA targeting CTCF verified by western blotting after 3 and 5 days of shRNA induction including shRNA non-silencing control (shNSC). Data shown are one representative of 3 independent experiments using immortalized HPECs. Percentage knockdown compared to shCTCF -Dox control, quantified by ImageJ. c Volcano plot of detected methylation changes in CTCF knockdown HPECE6/E7 after 5 days of dox exposure (cut-point, methylation Abs. Log2FC > 1.5, P

    Article Snippet: CTCF shRNA and controls were analyzed by western blotting using anti-CTCF rabbit monoclonal antibody (Cell Signaling #3418) to verify knockdown.

    Techniques: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation, shRNA, Western Blot

    HLA Class II chromatin landscapes of HLA-DR3 and DR15 haplotypes. a CTCF HiChIP looping interactions within the HLA Class II region reveal an insulated neighborhood that extends from BTNL2 to upstream of HLA-DQA2 . The COX-aligned DR3 haplotype is presented on top and the PGF-aligned DR15 haplotype is presented on the bottom. Gene and bp positions are presented specific for each haplotype. Orange arcs represent CTCF-mediated HiChIP interactions. Arc thickness is proportional to the frequency of observed paired-end tags (PETs) at each enhancer. b H3K27ac HiChIP looping interactions within the HLA Class II region reveal differential frequency and pattern in the HLA-DRB1 to HLA-DQB1 region between the HLA-DR15 and HLA-DR3 haplotypes. Black arcs represent H3K27ac-mediated HiChIP interactions. Significant p -values are presented for enhancers that exhibit differential binding frequencies (mean PETs) for one haplotype or the other and are colored by the haplotype that exhibits higher PET counts (HLA-DR3: blue; HLA-DR15: red). H3K27ac ChIP peaks are presented in blue (HLA-DR3) and red (HLA-DR15). Haplotype specific loops are indicated in blue (COX) or red (PGF). Corresponding haplotype specific enhancers are labeled as follows—COX: 1: 4.007–4.013 Mb, 2: 4.021–4.031 Mb, 3: 4.039–4.047 Mb, 4: 4.052–4.06 Mb, 5: 4.078–4.083 Mb; PGF: 1: 4.076–4.081 Mb, B : 4.092–4.102 Mb, C : 4.111–4.120 Mb, D : 4.124–4.129 Mb, E : 4.154–4.159 Mb. c Individual H3K27 HiChIP looping interactions for three subjects heterozygous for HLA-DR3 (left) and three subjects homozygous for HLA-DR15 (right). H3K27ac ChIP peaks, haplotype specific loops, and haplotype specific enhancers are presented in blue (HLA-DR3) and red (HLA-DR15)

    Journal: Nature Communications

    Article Title: Enhancer histone-QTLs are enriched on autoimmune risk haplotypes and influence gene expression within chromatin networks

    doi: 10.1038/s41467-018-05328-9

    Figure Lengend Snippet: HLA Class II chromatin landscapes of HLA-DR3 and DR15 haplotypes. a CTCF HiChIP looping interactions within the HLA Class II region reveal an insulated neighborhood that extends from BTNL2 to upstream of HLA-DQA2 . The COX-aligned DR3 haplotype is presented on top and the PGF-aligned DR15 haplotype is presented on the bottom. Gene and bp positions are presented specific for each haplotype. Orange arcs represent CTCF-mediated HiChIP interactions. Arc thickness is proportional to the frequency of observed paired-end tags (PETs) at each enhancer. b H3K27ac HiChIP looping interactions within the HLA Class II region reveal differential frequency and pattern in the HLA-DRB1 to HLA-DQB1 region between the HLA-DR15 and HLA-DR3 haplotypes. Black arcs represent H3K27ac-mediated HiChIP interactions. Significant p -values are presented for enhancers that exhibit differential binding frequencies (mean PETs) for one haplotype or the other and are colored by the haplotype that exhibits higher PET counts (HLA-DR3: blue; HLA-DR15: red). H3K27ac ChIP peaks are presented in blue (HLA-DR3) and red (HLA-DR15). Haplotype specific loops are indicated in blue (COX) or red (PGF). Corresponding haplotype specific enhancers are labeled as follows—COX: 1: 4.007–4.013 Mb, 2: 4.021–4.031 Mb, 3: 4.039–4.047 Mb, 4: 4.052–4.06 Mb, 5: 4.078–4.083 Mb; PGF: 1: 4.076–4.081 Mb, B : 4.092–4.102 Mb, C : 4.111–4.120 Mb, D : 4.124–4.129 Mb, E : 4.154–4.159 Mb. c Individual H3K27 HiChIP looping interactions for three subjects heterozygous for HLA-DR3 (left) and three subjects homozygous for HLA-DR15 (right). H3K27ac ChIP peaks, haplotype specific loops, and haplotype specific enhancers are presented in blue (HLA-DR3) and red (HLA-DR15)

    Article Snippet: Crosslinked protein/DNA was immunoprecipitated using antibodies to H3K27ac (rabbit polyclonal, Abcam #ab4729, 20ug/ml) and CTCF (rabbit mAb, clone D31H2, Cell Signaling #3418, 20ug/ml) and then purified by Protein A + G immunomagnetic beads.

    Techniques: HiChIP, Binding Assay, Positron Emission Tomography, Chromatin Immunoprecipitation, Labeling

    CTCF binds Cacna1b e37a locus in DRG-derived F11 cells and influences e37a splicing. ( A ) CTCF is expressed in F11 cells and is localized to cell nuclei. Fluorescence images show DAPI (nuclear maker), anti-CTCF, and overlay. ( B ) CTCF ChIP-qPCR targeted to Cacna1b e37 loci in F11 cells (Paired t-test, P value = 0.0274 for e37a vs e37b) (three biological replicates per condition). ( C ) Efficiency and specificity of qPCR primers. Cacna1b e36, e37a and e37b-specific primers amplify DNA with similar log-linear relationships between 1 × 10 −8 and 1.5 × 10 −1 ng cDNA ( upper panel ) (gray, pink and orange lineal regression lines for e36, e37a and e37b data sets have similar slopes; Fisher's tests P value = 0.4383). qPCR primer specificity was confirmed by using Cacna1b e37a and e37b cDNA clones ( lower panel ). ( D ) Western blot from F11 cells expressing control Gfp or Ctcf-Gfp cDNA 3 days after transfection. In control cells, anti-CTCF identifies a single band at ~135 kDa. Whereas, in cells overexpressing Ctcf-Gfp , anti-CTCF identifies two bands, endogenous ~135 kDa and a second ~160 kDa band consistent with the expected size of the CTCF-GFP fusion protein. Transfer membrane was cut at ~75 kDa (dotted red line) and the lower part treated with anti-GAPDH to measure GAPDH levels for protein expression and loading reference. CTCF protein levels relative to GAPDH (t-test P value = 0.0227 for Gfp vs Ctcf-Gfp ) (three biological replicates per condition). ( E ) qRT-PCR of Cacna1b e37a relative to e36 in F11 cells overexpressing Gfp or Ctcf -Gfp cDNA over three days (t-test P values = 0.2263 for Gfp vs Ctcf -Gfp for day 1; p=0.0065 for day 2; and p=0.0019 for day 3) (three biological replicates per condition). ( F ) Western blot from F11 cells expressing control siRNA ( Con ) or Ctcf siRNA 3 days after transfection. In control cells, anti-CTCF identifies endogenous CTCF at ~135 kDa, and reduced CTCF levels in cells expressing Ctcf siRNA. Transfer membrane was cut at ~75 kDa (dotted red line) and the lower part treated with anti-GAPDH to measure GAPDH levels for protein expression and loading reference. CTCF protein levels relative to GAPDH (t-test P value = 0.0008 for Con vs Ctcf siRNA) (three biological replicates per condition). ( G ) qRT-PCR of Cacna1b e37a relative to e36 in F11 cells expressing control siRNA ( Con ) or Ctcf siRNA over three days (t-test P values = 0.6035 for Con vs Ctcf siRNA for day 1; p=0.0022 for day 2; and p=0.0046 for day 3) (at least three biological replicates per condition). Biological replicates represent independent cell cultures, treatment and transfections.

    Journal: eLife

    Article Title: Cell-specific exon methylation and CTCF binding in neurons regulate calcium ion channel splicing and function

    doi: 10.7554/eLife.54879

    Figure Lengend Snippet: CTCF binds Cacna1b e37a locus in DRG-derived F11 cells and influences e37a splicing. ( A ) CTCF is expressed in F11 cells and is localized to cell nuclei. Fluorescence images show DAPI (nuclear maker), anti-CTCF, and overlay. ( B ) CTCF ChIP-qPCR targeted to Cacna1b e37 loci in F11 cells (Paired t-test, P value = 0.0274 for e37a vs e37b) (three biological replicates per condition). ( C ) Efficiency and specificity of qPCR primers. Cacna1b e36, e37a and e37b-specific primers amplify DNA with similar log-linear relationships between 1 × 10 −8 and 1.5 × 10 −1 ng cDNA ( upper panel ) (gray, pink and orange lineal regression lines for e36, e37a and e37b data sets have similar slopes; Fisher's tests P value = 0.4383). qPCR primer specificity was confirmed by using Cacna1b e37a and e37b cDNA clones ( lower panel ). ( D ) Western blot from F11 cells expressing control Gfp or Ctcf-Gfp cDNA 3 days after transfection. In control cells, anti-CTCF identifies a single band at ~135 kDa. Whereas, in cells overexpressing Ctcf-Gfp , anti-CTCF identifies two bands, endogenous ~135 kDa and a second ~160 kDa band consistent with the expected size of the CTCF-GFP fusion protein. Transfer membrane was cut at ~75 kDa (dotted red line) and the lower part treated with anti-GAPDH to measure GAPDH levels for protein expression and loading reference. CTCF protein levels relative to GAPDH (t-test P value = 0.0227 for Gfp vs Ctcf-Gfp ) (three biological replicates per condition). ( E ) qRT-PCR of Cacna1b e37a relative to e36 in F11 cells overexpressing Gfp or Ctcf -Gfp cDNA over three days (t-test P values = 0.2263 for Gfp vs Ctcf -Gfp for day 1; p=0.0065 for day 2; and p=0.0019 for day 3) (three biological replicates per condition). ( F ) Western blot from F11 cells expressing control siRNA ( Con ) or Ctcf siRNA 3 days after transfection. In control cells, anti-CTCF identifies endogenous CTCF at ~135 kDa, and reduced CTCF levels in cells expressing Ctcf siRNA. Transfer membrane was cut at ~75 kDa (dotted red line) and the lower part treated with anti-GAPDH to measure GAPDH levels for protein expression and loading reference. CTCF protein levels relative to GAPDH (t-test P value = 0.0008 for Con vs Ctcf siRNA) (three biological replicates per condition). ( G ) qRT-PCR of Cacna1b e37a relative to e36 in F11 cells expressing control siRNA ( Con ) or Ctcf siRNA over three days (t-test P values = 0.6035 for Con vs Ctcf siRNA for day 1; p=0.0022 for day 2; and p=0.0046 for day 3) (at least three biological replicates per condition). Biological replicates represent independent cell cultures, treatment and transfections.

    Article Snippet: Membranes were cut at ~75 kDa and > 75 kDa region incubated in CTCF primary antibody (CTCF (D31H2) XP Rabbit mAb - Cell Signaling Technology, Cat# 3418, RRID: AB_2086791 ) and < 75 kDa region with GAPDH primary antibody (GAPDH (14C10) Rabbit mAb - Cell Signaling Technology, Cat# 2118, RRID: AB_561053 ).

    Techniques: Derivative Assay, Fluorescence, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Clone Assay, Western Blot, Expressing, Transfection, T-Test, Quantitative RT-PCR

    A 26-bp deletion of the Prdm9 gene leads to production of an N-terminal truncated PRDM9 protein. a Schematic representation of the PRDM9 proteins expressed from the wild-type B6 allele and the CBA ΔKB1 allele of the Prdm9 gene. These two alleles have distinct zinc finger arrays. b Immunoblot of PRDM9 expression in Prdm9 −/− , Prdm9 ΔKB1/ΔKB1 , Prdm9 +/+ (B6), and Prdm9 ΔKB1/+ (heterozygous Prdm9 ΔKB1 / Prdm9 Dom2 from B6) testes at 13 dpp. Each lane was loaded with 100 μg of testis protein extracts. The same membrane was stained with an anti-CTCF antibody as loading control. The predicted molecular weights of PRDM9 B6 and PRDM9ΔKB1 are 97 and 95 kDa, respectively. Both proteins migrate faster than predicted. The right panel reports signal intensities in arbitrary units of each band (PRDM9, CTCF and the top and bottom PRDM9 bands in ΔKB1/+) and the ratio of intensities between PRDM9 and CTCF (PRDM9/CTCF). c Immunofluorescent analysis of PRDM9, SYCP3 (meiotic chromosomes axial elements marker), and γH2AX (DSB formation marker) expression in chromosome spreads of Prdm9 +/+ (B6), Prdm9 ΔKB1/+ (heterozygous Prdm9 ΔKB1 / Prdm9 Dom2 from B6), Prdm9 ΔKB1/ΔKB1 , and Prdm9 −/− spermatocytes at leptonema. Within one genotype, intensity of γH2AX can vary at leptonema and we did not detect consistent differences between genotypes. Chromosome spreads were prepared from 10-week-old ( Prdm9 +/+ , Prdm9 ΔKB1/+ , and Prdm9 ΔKB1/ΔKB1 ) or 5-week-old ( Prdm9 −/− ) mice. Scale bars = 10 μm

    Journal: Chromosoma

    Article Title: The PRDM9 KRAB domain is required for meiosis and involved in protein interactions

    doi: 10.1007/s00412-017-0631-z

    Figure Lengend Snippet: A 26-bp deletion of the Prdm9 gene leads to production of an N-terminal truncated PRDM9 protein. a Schematic representation of the PRDM9 proteins expressed from the wild-type B6 allele and the CBA ΔKB1 allele of the Prdm9 gene. These two alleles have distinct zinc finger arrays. b Immunoblot of PRDM9 expression in Prdm9 −/− , Prdm9 ΔKB1/ΔKB1 , Prdm9 +/+ (B6), and Prdm9 ΔKB1/+ (heterozygous Prdm9 ΔKB1 / Prdm9 Dom2 from B6) testes at 13 dpp. Each lane was loaded with 100 μg of testis protein extracts. The same membrane was stained with an anti-CTCF antibody as loading control. The predicted molecular weights of PRDM9 B6 and PRDM9ΔKB1 are 97 and 95 kDa, respectively. Both proteins migrate faster than predicted. The right panel reports signal intensities in arbitrary units of each band (PRDM9, CTCF and the top and bottom PRDM9 bands in ΔKB1/+) and the ratio of intensities between PRDM9 and CTCF (PRDM9/CTCF). c Immunofluorescent analysis of PRDM9, SYCP3 (meiotic chromosomes axial elements marker), and γH2AX (DSB formation marker) expression in chromosome spreads of Prdm9 +/+ (B6), Prdm9 ΔKB1/+ (heterozygous Prdm9 ΔKB1 / Prdm9 Dom2 from B6), Prdm9 ΔKB1/ΔKB1 , and Prdm9 −/− spermatocytes at leptonema. Within one genotype, intensity of γH2AX can vary at leptonema and we did not detect consistent differences between genotypes. Chromosome spreads were prepared from 10-week-old ( Prdm9 +/+ , Prdm9 ΔKB1/+ , and Prdm9 ΔKB1/ΔKB1 ) or 5-week-old ( Prdm9 −/− ) mice. Scale bars = 10 μm

    Article Snippet: The following primary antibodies and dilutions were used: rabbit anti-PRDM9 polyclonal (Grey et al. ), 1:500; rabbit anti-CTCF monoclonal (Cell Signaling Technology, D31H2), 1:2000; rabbit anti-CXXC1 polyclonal (Millipore, ABE211), 1:5000; rat anti-tubulin α monoclonal (Abcam, ab6161), 1:3000; rabbit anti-PIH1D1 polyclonal (Proteintech, AP19427), 1:5000; rabbit anti-CEP70 polyclonal (Abnova, PAB17658), 1:1000; mouse anti-MCRS1 polyclonal (Abnova, H00010445-B01P), 1:250; anti-Gal4 AD (Millipore, 06-283), 1:3000; and anti-Gal4 BD (Sigma, G3042), 1:2000.

    Techniques: Crocin Bleaching Assay, Expressing, Staining, Marker, Mouse Assay