Revision 5

#30559Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IF-IC, ChIP, ChIP-seq, C&R

REACTIVITY:

H M Mk

SENSITIVITY:

Endogenous

MW (kDa):

78

Source/Isotype:

Rabbit IgG

UniProt ID:

#O00257

Entrez-Gene Id:

8535

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Immunocytochemistry) 1:200
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

CBX4 (E6L7X) Rabbit mAb recognizes endogenous levels of total CBX4 protein.

Species Reactivity:

Human, Mouse, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro166 of human CBX4 protein.

Background

The polycomb group (PcG) proteins contribute to the maintenance of cell identity, stem cell self-renewal, cell cycle regulation, and oncogenesis by maintaining the silenced state of genes that promote cell lineage specification, cell death, and cell-cycle arrest (1-4). PcG proteins exist in two complexes that cooperate to maintain long-term gene silencing through epigenetic chromatin modifications. The first complex, EED-EZH2, is recruited to genes by DNA-binding transcription factors and methylates histone H3 on Lys27. This histone methyl-transferase activity requires the Ezh2, Eed, and Suz12 subunits of the complex (5). Histone H3 methylation at Lys27 facilitates the recruitment of the second complex, PRC1, which ubiquitinylates histone H2A on Lys119 (6). CBX4 is a component of the PRC1 complex, which together with Ring1 strongly enhances the E3 ubiquitin ligase activity of the Ring2 catalytic subunit (7,8). CBX4 itself is a SUMO E3 ligase, and its function influences EMT, DNA damage response, tumor angiogenesis, and self-renewal (9-13).

  1. Boyer, L.A. et al. (2006) Nature 441, 349-53.
  2. Lee, T.I. et al. (2006) Cell 125, 301-13.
  3. Cao, R. et al. (2002) Science 298, 1039-43.
  4. Müller, J. et al. (2002) Cell 111, 197-208.
  5. Cao, R. and Zhang, Y. (2004) Mol Cell 15, 57-67.
  6. Wang, H. et al. (2004) Nature 431, 873-8.
  7. Satijn, D.P. et al. (1997) Mol Cell Biol 17, 6076-86.
  8. Cao, R. et al. (2005) Mol Cell 20, 845-54.
  9. Kagey, M.H. et al. (2003) Cell 113, 127-37.
  10. Li, B. et al. (2007) Biochem J 405, 369-78.
  11. Ismail, I.H. et al. (2012) Nucleic Acids Res 40, 5497-510.
  12. Li, J. et al. (2014) Cancer Cell 25, 118-31.
  13. Klauke, K. et al. (2013) Nat Cell Biol 15, 353-62.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 5
#30559

CBX4 (E6L7X) Rabbit mAb

Western Blotting Image 1: CBX4 (E6L7X) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using CBX4 (E6L7X) Rabbit mAb. As expected, HDLM2 and NTERA-2 cells do not highly express CBX4.
Immunoprecipitation Image 1: CBX4 (E6L7X) Rabbit mAb Expand Image
Immunoprecipitation of CBX4 from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CBX4 (E6L7X) Rabbit mAb. Western blot analysis was performed using CBX4 (E6L7X) Rabbit mAb.
Immunofluorescence Image 1: CBX4 (E6L7X) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of Caki-1 cells using CBX4 (E6L7X) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Chromatin Immunoprecipitation Image 1: CBX4 (E6L7X) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either CBX4 (E6L7X) Rabbit mAb or RING1B (D22F2) XP® Rabbit mAb #5694, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across HoxA, a known target gene of CBX4 and RING1B (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: CBX4 (E6L7X) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either CBX4 (E6L7X) Rabbit mAb or RING1B (D22F2) XP® Rabbit mAb #5694, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figures show binding across HoxA (upper) and HoxD (lower), known target genes of CBX4 and RING1B (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: CBX4 (E6L7X) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either CBX4 (E6L7X) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517 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.
CUT and RUN Image 1: CBX4 (E6L7X) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either CBX4 (E6L7X) Rabbit mAb or Bmi1 (D20B7) XP® Rabbit mAb #6964, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across HoxA1 gene, a known target gene of CBX4 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: CBX4 (E6L7X) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either CBX4 (E6L7X) Rabbit mAb or Bmi1 (D20B7) XP® Rabbit mAb #6964, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across HoxA (upper) and HoxD (lower), known target genes of CBX4 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: CBX4 (E6L7X) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either CBX4 (E6L7X) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, 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.