Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Human

Cited:

Human, Mouse

Applications

Validated:

Western Blot, ELISA, Flow Cytometry, CyTOF-ready

Cited:

Western Blot, Flow Cytometry, Immunoprecipitation, STED Microscopy

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
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Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human CMG‑2/ANTXR2 isoform 1
Gln34-Asn317
Accession # P58335

Specificity

Detects human CMG‑2/ANTXR2 in direct ELISAs and Western blots. In direct ELISAs and Western blots, approximately 20% cross-reactivity with recombinant mouse CMG-2 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human CMG‑2/ANTXR2 Antibody

Detection of CMG‑2/ANTXR2 antibody in Human Monocytes antibody by Flow Cytometry.

Detection of CMG‑2/ANTXR2 in Human Monocytes by Flow Cytometry.

Human whole blood monocytes were stained with Human CMG-2/ANTXR2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2940, filled histogram) or control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107).

Detection of Human CMG-2/ANTXR2 by Western Blot

Detection of Human CMG-2/ANTXR2 by Western Blot

Number and localization of glycan sidechains determine trafficking efficiency of TEM8 and CMG2.A) Endoglycosidase H (EndoH) treatment on TEM8 and CMG2 single mutants. HeLa cells were transfected for 48h with the respective cDNAs. 40 μg of cell extracts were treated or not with EndoH as described before. Samples were analyzed by SDS-PAGE and Western Blotting. B) Quantification of surface biotinylation experiments to determine amount of TEM8 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 C) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against TEM8-HA. D) Quantification of surface biotinylation experiments to determine amount of CMG2 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 E) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against CMG2-V5. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human CMG-2/ANTXR2 by Western Blot

Detection of Human CMG-2/ANTXR2 by Western Blot

Number and localization of glycan sidechains determine trafficking efficiency of TEM8 and CMG2.A) Endoglycosidase H (EndoH) treatment on TEM8 and CMG2 single mutants. HeLa cells were transfected for 48h with the respective cDNAs. 40 μg of cell extracts were treated or not with EndoH as described before. Samples were analyzed by SDS-PAGE and Western Blotting. B) Quantification of surface biotinylation experiments to determine amount of TEM8 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 C) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against TEM8-HA. D) Quantification of surface biotinylation experiments to determine amount of CMG2 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 E) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against CMG2-V5. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human CMG-2/ANTXR2 by Immunoprecipitation

Detection of Human CMG-2/ANTXR2 by Immunoprecipitation

Glycosylation acts as a buffer for CMG2 ectodomain mutations.A) Graphic showing the disulfide bridge C39-C218 (blue) present in CMG2 WT B) Fibroblast cells were treated or not with tunicamycin for 16h and TCE were analyzed by SDS-PAGE and Western Blot. Representative Western Blot with control fibroblasts and patient fibroblasts. Calnexin serves as a loading control. C) Quantification of total protein levels. CMG2 levels are normalized to WT protein level without tunicamycin treatment, which was set to 100%. Statistics were calculated using an unpaired t-test. Errors represent standard deviation. n ≥ 3. * p≤0.05 Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human CMG-2/ANTXR2 by Immunoprecipitation

Detection of Human CMG-2/ANTXR2 by Immunoprecipitation

Loss of glycosylation affects binding of Anthrax toxin to TEM8 but not to CMG2.A and C) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated for 1h at 4°C with 500 ng/ml PA83 and shifted to 37°C for 10 min to induce cleavage and heptamerization. Immunoprecipitates against TEM8-HA/CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against PA and TEM8-HA/CMG2-V5. Control cells are non-transfected. D50A is a binding deficient mutant that serves as a negative control. B and D) HeLa cells were transfected for 48h with the respective cDNAs. Cells were lysed and incubated for 1h at 4°C with 1 μg/ml PA83. Immunoprecipitates against TEM8-HA/CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against PA and TEM8-HA/CMG2-V5. Control cells are non-transfected. D50A is a binding deficient mutant that serves as a negative control. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human CMG-2/ANTXR2 by Immunoprecipitation

Detection of Human CMG-2/ANTXR2 by Immunoprecipitation

CMG2 and TEM8 can undergo N-glycosylation on all predicted sites.A) Graphics depicting glycosylation sites on TEM8 and CMG2. Sites in red are unique to the respective proteins, N260 in CMG2 (yellow) corresponds to N262 in TEM8. B) Expression of TEM8 glycosylation mutants in HeLa cells. Cells were transfected for 48h with the respective cDNAs. Expression was analyzed by SDS-PAGE and Western Blotting. C) Expression of all CMG2 glycosylation mutants in HeLa cells. Cells were transfected for 48h with the respective cDNAs. Expression was analyzed by SDS-PAGE and Western Blotting. D) Endoglycosidase F (NGaseF) treatment on TEM8 glycosylation mutants. 40 μg of cell extracts were treated or not with NGaseF and analyzed by SDS-PAGE and Western Blotting. E) Endoglycosidase F (NGaseF) treatment on CMG2 glycosylation mutants. 40 μg of cell extracts were treated or not with NGaseF and analyzed by SDS-PAGE and Western Blotting. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human CMG-2/ANTXR2 by Western Blot

Detection of Human CMG-2/ANTXR2 by Western Blot

Non-glycosylated TEM8 is an ER quality control and ERAD substrate.A) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated or not with MG132, an inhibitor of the proteasome or Bafilomycin A1, a drug preventing endosomal acidification and thus lysosomal degradation. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and TEM8-HA. B) HEK cells stably expressing CMG2 under the control of a tetracycline inducible promotor were induced for 24h with 0.1μg/ml doxycycline. Cells were treated or not with MG132 or Bafilomycin A1. Immunoprecipitates against CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and CMG2-V5. C) HeLa cells were treated or not with tunicamycin, an antibiotic blocking the co-translational transfer of glycan sidechains in the ER by blocking the oligosaccharyltransferase (OST) for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for TEM8 or Calnexin as a negative control. D) RpeI cells were treated or not with tunicamycin for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for CMG2 or Calnexin as a negative control. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25781883), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human CMG‑2/ANTXR2 Antibody

Application
Recommended Usage

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

ELISA

This antibody functions as an ELISA detection antibody when paired with Mouse Anti-Human CMG‑2/ANTXR2 Monoclonal Antibody (Catalog # MAB29401).

This product is intended for assay development on various assay platforms requiring antibody pairs.

Flow Cytometry

2.5 µg/106 cells
Sample: Human whole blood monocytes

Western Blot

0.1 µg/mL
Sample: Recombinant Human CMG‑2/ANTXR2 (Catalog # 2940-CM)

Flow Cytometry Panel Builder

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Save time and reduce costly mistakes by quickly finding compatible reagents using the Panel Builder Tool.

Advanced Features

  • Spectra Viewer - Custom analysis of spectra from multiple fluorochromes
  • Spillover Popups - Visualize the spectra of individual fluorochromes
  • Antigen Density Selector - Match fluorochrome brightness with antigen density
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Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.


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Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: CMG-2/ANTXR2

Capillary Morphogenesis Gene-2 (CMG-2) is a widely expressed anthrax toxin receptor (ATR) family protein (1‑3). CMG-2 is a 55 kDa type I transmembrane (TM) protein that contains a 33 amino acid (aa) signal sequence, a 284 aa extracellular domain (ECD), a 24 aa TM segment, and a 147 aa cytoplasmic domain. There are three additional isoforms. Isoforms 4 shows a 12 aa insertion in the cytoplasmic region; isoform 2 shows a 103 aa deletion in the ECD; and isoform 3 is a truncated, 20 kDa, 289 aa soluble form. The main functional domain of CMG-2 is an extracellular integrin-like von Willebrand factor type A (VWA) domain with a metal ion dependent adhesion site (MIDAS). This domain adheres selectively to collagen type IV and laminin (1‑5). CMG-2 isoform 2 is induced in HUVEC as they undergo capillary formation in collagen matrices in vitro (3). CMG-2 is mutated in juvenile hyaline fibromatosis and infantile systemic hyalinosis disorders, and several of these mutations result in loss of laminin binding (6). CMG-2 and the related protein ATR/TEM8 serve as receptors for the protective antigen (PA) of Bacillus Anthracis (1, 2). After binding the VWA domain, PA undergoes furin-type cleavage, forms a heptameric receptor/PA pre-pore and binds LF or EF toxin subunits (5, 7, 8). Transport to low pH endosomes, which requires CMG-2 ubiquitination and interaction with the LDL receptor related protein LRP6 (9, 10), allows PA pore formation and release of toxin to the cytoplasm (10, 11). Soluble CMG-2 VWA domain acts as a dummy receptor that can protect cultured cells from anthrax intoxication (2). Within the extracellular region, human CMG-2 shares 84%, 81%,89% and 93% amino acid sequence homology with mouse, rat, bovine, and canine CMG-2, respectively. CMG-2 VWA domain also shares 60% aa identity with ATR/TEM8.

References

  1. Scobie, H.M. and J.A.T. Young (2005) Curr. Opin. Microbiol. 8:106.
  2. Scobie, H.M. et al. (2003) Proc. Natl. Acad. Sci. USA 100:5170.
  3. Bell, S.E. et al. (2001) J. Cell Sci. 114:2755.
  4. Lacy, D.B. et al. (2004) Proc. Natl. Acad. Sci. USA 101:6367.
  5. Santelli, E. et al. (2004) Nature 430:905.
  6. Dowling, O. et al. (2003) Am. J. Hum. Genet. 73:957.
  7. Wigelsworth, D.J. et al. (2004) J. Biol. Chem. 279:23349.
  8. Go, M.Y. et al. (2006) J. Mol. Biol. 360:145.
  9. Abrami, L. et al. (2006) J. Cell Biol. 172:309.
  10. Wei, W. et al. (2006) Cell 124:1141.
  11. Lacy, D.B. et al. (2004) Proc. Natl. Acad. Sci. USA 101:13147.

Long Name

Capillary Morphogenesis Protein 2

Alternate Names

ANTXR2, cI-35, CMG2, ISH, JHS

Entrez Gene IDs

118429 (Human); 71914 (Mouse)

Gene Symbol

ANTXR2

UniProt

Additional CMG-2/ANTXR2 Products

Product Documents for Human CMG‑2/ANTXR2 Antibody

Certificate of Analysis

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Product Specific Notices for Human CMG‑2/ANTXR2 Antibody

For research use only

Related Research Areas

Citations for Human CMG‑2/ANTXR2 Antibody

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Protocols

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