Revision 5

#25069Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) orders@cellsignal.com

Support: 877-678-TECH (8324)

Web: info@cellsignal.com cellsignal.com

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

WB, IP, ChIP, ChIP-seq, C&R, C&T

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

70

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q14781

Entrez-Gene Id:

84733

Product Information

Product Usage Information

For optimal ChIP results, use 5 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.

The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Chromatin IP 1:100
Chromatin IP-seq 1:100
CUT&RUN 1:50
CUT&Tag 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

CBX2 (E3N6A) Rabbit mAb recognizes endogenous levels of total CBX2 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala100 of human CBX2 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). Chromobox protein homolog 2 (CBX2) is a core component of the PRC1 complex, which binds to the H3K27me3 mark and recruits PRC1 for transcriptional repression (7). CBX2 is frequently overexpressed in various cancers, including breast, ovarian, and AR-independent prostate cancer, where it is associated with poor prognosis (8-14). Furthermore, elevated CBX2 expression levels correlate with larger tumor size, increased lymph node metastasis, and positive human epidermal growth factor receptor 2 (HER-2) status in breast cancer (8). Mutations in CBX2 have also been attributed to 46,XY disorders of sex development (DSD), at least partially due to misregulation of the NR5A1/SF1 gene (15,16).

  1. Boyer, L.A. et al. (2006) Nature 441, 349-53.
  2. Lee, T.I. et al. (2006) Cell 125, 301-13.
  3. Park, I.K. et al. (2003) Nature 423, 302-5.
  4. Molofsky, A.V. et al. (2003) Nature 425, 962-7.
  5. Tavares, L. et al. (2012) Cell 148, 664-78.
  6. Gao, Z. et al. (2012) Mol Cell 45, 344-56.
  7. Kaustov, L. et al. (2011) J Biol Chem 286, 521-9.
  8. Chen, W.Y. et al. (2017) Oncol Lett 13, 1149-1156.
  9. Liang, Y.K. et al. (2017) Oncotarget 8, 92375-92387.
  10. Piqué, D.G. et al. (2019) Br J Cancer 120, 746-753.
  11. Zheng, S. et al. (2019) Am J Transl Res 11, 1668-1682.
  12. Iqbal, M.A. et al. (2021) Mol Oncol 15, 1450-1465.
  13. Wheeler, L.J. et al. (2018) Oncogenesis 7, 92.
  14. Clermont, P.L. et al. (2016) Clin Epigenetics 8, 16.
  15. Biason-Lauber, A. et al. (2009) Am J Hum Genet 84, 658-63.
  16. Sproll, P. et al. (2019) Sci Rep 9, 16552.

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 ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag

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.
SimpleChIP is a registered 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
#25069

CBX2 (E3N6A) Rabbit mAb

Western Blotting Image 1: CBX2 (E3N6A) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using CBX2 (E3N6A) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Low expression of CBX2 protein in AsPC-1 cells is consistent with the predicted expression pattern.
Western Blotting Image 2: CBX2 (E3N6A) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using CBX2 (E3N6A) Rabbit mAb.
Immunoprecipitation Image 1: CBX2 (E3N6A) Rabbit mAb Expand Image
Immunoprecipitation of CBX2 protein from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CBX2 (E3N6A) Rabbit mAb. Western blot analysis was performed using CBX2 (E3N6A) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: CBX2 (E3N6A) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either CBX2 (E3N6A) Rabbit mAb, CBX2 Antibody #18687, 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 Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across HoxA1, a known target gene of CBX2 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: CBX2 (E3N6A) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either CBX2 (E3N6A) Rabbit mAb, CBX2 Antibody #18687, 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 Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across HoxA (upper) and HoxD (lower), known target genes of CBX2 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: CBX2 (E3N6A) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either CBX2 (E3N6A) 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: CBX2 (E3N6A) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and CBX2 (E3N6A) Rabbit mAb using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across TAL1, a known target gene of CBX2 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: CBX2 (E3N6A) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and CBX2 (E3N6A) Rabbit mAb using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including TAL1 (lower), a known target gene of CBX2 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: CBX2 (E3N6A) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either CBX2 (E3N6A) 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 human TAL1 promoter primers, human FOXP2 promoter primers, and human PRSS1 upstream primers. 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 Tag Image 1: CBX2 (E3N6A) Rabbit mAb Expand Image
CUT&Tag was performed with NCCIT cells and CBX2 (E3N6A) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across HOXA1, a known target gene of CBX2 (see our ChIP-qPCR figure).
CUT and Tag Image 2: CBX2 (E3N6A) Rabbit mAb Expand Image
CUT&Tag was performed with NCCIT cells and CBX2 (E3N6A) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across the HOXA gene cluser (upper), and the HOXD gene cluster (lower), which are known target genes of CBX2 (see our ChIP-qPCR figure).