Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Chromatin Regulation / Epigenetics

CTCF (D1A7) XP® Rabbit mAb #3417

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC ChIP H R Mk (B) Endogenous 140 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  R=Rat  Mk=Monkey  B=Bovine
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

CTCF (D1A7) XP® Rabbit mAb detects endogenous levels of total CTCF protein. This antibody does not cross-react with BORIS.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the human CTCF protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using CTCF (D1A7) XP® Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using CTCF (D1A7) XP® Rabbit mAb (green). Actin filaments are labeled with DY-554 phalloidin (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of CTCF (D1A7) XP® Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human c-Myc promoter primers, SimpleChIP® Human H19/Igf2 ICR Primers #5172, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.


Background

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).

  1. Klenova, E.M. et al. (2002) Semin Cancer Biol 12, 399-414.
  2. Klenova, E.M. et al. (1993) Mol Cell Biol 13, 7612-24.
  3. Loukinov, D.I. et al. (2002) Proc Natl Acad Sci USA 99, 6806-11.
  4. Mukhopadhyay, R. et al. (2004) Genome Res 14, 1594-602.
  5. Hark, A.T. et al. (2000) Nature 405, 486-9.
  6. Ohta, T. et al. (1999) Am J Hum Genet 64, 397-413.
  7. Chao, W. et al. (2002) Science 295, 345-7.
  8. Yu, W. et al. (2004) Nat Genet 36, 1105-10.
  9. El-Kady, A. and Klenova, E. (2005) FEBS Lett 579, 1424-34.
  10. Filippova, G.N. et al. (1998) Genes Chromosomes Cancer 22, 26-36.
  11. Filippova, G.N. et al. (2002) Cancer Res 62, 48-52.
  12. Vatolin, S. et al. (2005) Cancer Res 65, 7751-62.
  13. Hong, J.A. et al. (2005) Cancer Res 65, 7763-74.
  14. D'Arcy, V. et al. (2008) Br J Cancer 98, 571-9.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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