DDR2 Antibody [Unconjugated]

Simple Western lane view shows lysates of HEK293 human embryonic kidney cell line transfected with human DDR2 untreated (-) or treated (+) with Calyculin A for 10 minutes, loaded at 0.2 mg/mL. A specific band was detected for DDR2 at approximately 139 kDa (as indicated) using 2.5 µg/mL of Goat Anti-Human DDR2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2538) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the12-230 kDa separation system.

Role of DDR2 in MT1-MMP-dependent collagen film degradation and collagen invasion.A, human RASF or HDF transfected with siNT, siRNA for DDR2 (siDDR2), or siRNA for beta 1 integrin (siITGB1) were subjected to collagen film degradation assay as described under “Experimental Procedures” (left panel). Conditioned media from this assay were analyzed by zymography. P, pro-MMP-2; A, active MMP-2. Scale bars, 270 μm. B, human RASF transfected with siNT, siRNA for DDR2, or siRNA for ITGB1 were subjected to Transwell collagen invasion assay as described under “Experimental Procedures” (upper panel). Levels of DDR2, ITGB1, and actin in transfected cells were analyzed by Western blotting (bottom panel). These are combined data of three independent experiments (n = 6 for each experiments). Error bars represent S.E. ***, p > 0.001; n.s., not significant. Image collected and cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

DDR2 but not integrins mediates collagen signaling to activate MT1-MMP functions in RASF.A, human RASF were stimulated with collagen I (Col-I) in the presence or absence of beta 1 integrin-inhibitory antibody (6S6) or -activating antibody (P4G11) for 24 h in serum-free medium. Conditioned media and cell lysates were analyzed as in A. B, cell lysates from HT1080 human fibrosarcoma cells, RASF, and HDF were analyzed for DDR1 and DDR2 expression by Western blotting. RASF and HDF express DDR2 but not DDR1, whereas HT1080 expresses both DDRs. C, RASF were transfected with siRNA for DDR2 (siDDR2), beta 1 integrin (siITGB1), and/or siNT as indicated. 48 h later, cell lysates were subjected to Western blotting analyses for DDR2, ITGB1, and actin (left panel). 48 h after siRNA transfection, cells were also stimulated with collagen I (100 μg/ml) and cultured for a further 72 h. siNT-transfected cells were also treated with dasatinib (Dasa; 100 nm). Conditioned media and cell lysates were analyzed as in A (right panel). D, RASF transfected with siNT, siRNA for DDR2, or siRNA for ITGB1 were stimulated with collagen II (Col-II) of human origin (100 μg/ml) and bovine origin (100 μg/ml) for 48 h. Conditioned media and cell lysates were analyzed as in A. E, RASF transfected with siNT, siRNA for DDR2, or siRNA for ITGB1 were subjected to F-gelatin film degradation assay as in D. Scale bars, 270 μm. P, pro-MMP-2; A, active MMP-2; Zymo, zymography. Image collected and cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

DDR2 mediates collagen signaling to activate MT1-MMP functions in HDF.A, HDF were transfected with siRNA for DDR2 (siDDR2), siRNA for beta 1 integrin (siITGB1), and/or siNT as indicated. 48 h later, cell lysates were subjected to Western blotting analyses for DDR2, ITGB1, and actin (right panel).48 h after siRNA transfection, cells were further stimulated with collagen I (Col-I) (100 μg/ml) and cultured for a further 48 h. Conditioned media were analyzed by zymography. B, HDF transfected with siNT, siRNA for DDR2, or siRNA for ITGB1 were subjected to F-gelatin film degradation assay as in Fig. 2E. Scale bars, 270 μm. P, pro-MMP-2; A, active MMP-2. Image collected and cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

Role of DDR2 in collagen-induced MT1-MMP gene expression.A, human RASF were stimulated with or without collagen I (Col-I) (open bars or closed bars, respectively) for 24 or 48 h, and the level of MT1-MMP gene expression was examined by qPCR (n = 6). ***, p > 0.001. B, RASF transfected with siNT, siRNA for DDR2 (siDDR2), or siRNA for beta 1 integrin (siITGB1) were stimulated with collagen (open bars). Error bars represent S.E. (n = 6). ***, p > 0.001; **, p > 0.01; n.s., not significant. C, RASF transfected with siNT, siRNA for DDR2, or siRNA for ITGB1 were stimulated with collagen I (100 μg/ml) in the presence or absence of GM6001for 72 h. Cell lysates were subjected to Western blotting analyses for MT1-MMP, actin, DDR2, and ITGB1. Cont, control. Image collected and cropped by CiteAb from the following publication (//linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

DDR2 receptor phosphorylation and in vitro kinase assay(A) Plots of relative DDR2 phosphorylation levels as a function of time after collagen stimulation. Temporal profiles of DDR2 sites show distinct responses to collagen stimulation. Measurements are expressed relative to the 24 h time point. (B) In vitro kinase assay measuring the incorporation of 32P into the Axltide substrate peptide. Equal amounts (750 nM) of Src and DDR2 were mixed together with 300 pM TDA in kinase assay buffer. Data represent the fold change relative to control (DDR2). (C) Immunoblot of total phosphotyrosine levels (using the 4G10 antibody) obtained from the in vitro kinase reaction showing increased phosphorylation levels that correlate with DDR2 kinase activity. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.

DDR2 phosphorylates SHP-2 in a temporal and kinase-dependent manner(A) SRM analysis of DDR2 activation loop (Tyr736 and Tyr740/Tyr741) and SHP-2 (Tyr62) phosphorylation at 0 and 24 h post-stimulation with collagen I (n=3). Values are means±S.E.M., **P<0.01 using paired Student's t test. Representative transitions are shown as indicated in Supplementary Table S3 (at //www.biochemj.org/bj/454/bj4540501add.htm). (B) Immunoblot of DDR2 and SHP-2 (Tyr542) phosphorylation shows a temporal up-regulation of SHP-2 phosphorylation upon simulation with collagen. Phosphotyrosine (pY) is measured by the 4G10 antibody. (C) Immunoblot of kinase-dead DDR2 mutants (K608M and K608E) indicate that tyrosine phosphorylation of SHP-2 (Tyr542) is dependent on DDR2 kinase activity. EV, empty vector control. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.

DDR2 phosphorylates SHP-2 in a temporal and kinase-dependent manner(A) SRM analysis of DDR2 activation loop (Tyr736 and Tyr740/Tyr741) and SHP-2 (Tyr62) phosphorylation at 0 and 24 h post-stimulation with collagen I (n=3). Values are means±S.E.M., **P<0.01 using paired Student's t test. Representative transitions are shown as indicated in Supplementary Table S3 (at //www.biochemj.org/bj/454/bj4540501add.htm). (B) Immunoblot of DDR2 and SHP-2 (Tyr542) phosphorylation shows a temporal up-regulation of SHP-2 phosphorylation upon simulation with collagen. Phosphotyrosine (pY) is measured by the 4G10 antibody. (C) Immunoblot of kinase-dead DDR2 mutants (K608M and K608E) indicate that tyrosine phosphorylation of SHP-2 (Tyr542) is dependent on DDR2 kinase activity. EV, empty vector control. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.

Targeted proteomic profiling of lung SCC DDR2 mutants(A) Domain organization and location of DDR2 point mutations used in the present study. JM, juxtamembrane; TM, transmembrane domain. (B) Colony formation assay of DDR2 mutants grown in 3D collagen I gels (n=3). Values are means±S.E.M., statistical significance of wild-type and mutant DDR2 data compared with the empty vector control was performed by ANOVA with Dunnett's post-test where **P<0.01 and *P<0.05. (C) Immunoblot of mutant DDR2 cells after stimulation with collagen I for 24 h. Phosphotyrosine (pY) is measured by the pY100 antibody. (D) Normalized densitometry measurements of phosphotyrosine and phosphorylated SHP-2 blots (n=3). ***P<0.001 and **P<0.01. Normalization is performed relative to the loading control, tubulin. (E) SRM analysis of DDR2 receptor and SHP-2 phosphorylation in wild-type and lung SCC point mutations post activation with collagen I at 24 h (n=3). Values are means±S.E.M., statistical significance of mutant DDR2 phosphorylation data compared with wild-type DDR2 was performed by paired Student's t test where ***P<0.001 and *P<0.05. Representative transitions are shown as indicated in Supplementary Table S3 at //www.biochemj.org/bj/454/bj4540501add.htm. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, Simple Western, IHC
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

DDR2 Antibody [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant human DDR2
  Gln24-Arg399
  Accession # Q16832
• Specificity: Detects human DDR2 in direct ELISAs and Western blots. In Western blots, approximately 5% cross-reactivity with recombinant human DDR1 is observed.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: DDR2
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • Immunohistochemistry 5-15 ug/mL
  • Simple Western 2.5 ug/mL
  • Western Blot 0.1 ug/mL
• Application Notes: In Simple Western only 10-15 uL of the recommended dilution is used per data point.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  12 months from date of receipt, -20 to -70 degreesC as supplied. 1 month, 2 to 8 degreesC under sterile conditions after reconstitution. 6 months, -20 to -70 degreesC under sterile conditions after reconstitution.
• Buffer: 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.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for DDR2 Antibody [Unconjugated]

• CD167 antigen-like family member B
• CD167b antigen
• DDR2
• Discoidin domain receptor 2
• discoidin domain receptor family, member 2
• discoidin domain receptor tyrosine kinase 2
• discoidin domain-containing receptor 2
• EC 2.7.10
• EC 2.7.10.1
• hydroxyaryl-protein kinase
• migration-inducing gene 16 protein
• neurotrophic tyrosine kinase receptor related 3
• Neurotrophic tyrosine kinase, receptor-related 3
• NTRKR3cell migration-inducing protein 20
• Receptor protein-tyrosine kinase TKT
• TKT
• TKTMIG20a
• Trk3
• TYRO10
• Tyro-10
• Tyrosine-protein kinase TYRO10
• tyrosylprotein kinase

Background

DDR2, also known as TYR010 and TKT, is a widely expressed 130 kDa type I transmembrane glycoprotein belonging to the discoidin-like domain containing subfamily of receptor tyrosine kinases (1). Mature human DDR2 consists of a 378 amino acid (aa) extracellular domain (ECD) that includes the discoidin-like domain, a 22 aa transmembrane segment, and a 434 aa cytoplasmic domain that includes the kinase domain (2). Within the ECD, human DDR2 shares 53% aa sequence identity with DDR1 and 97% aa sequence identity with mouse DDR2. The discoidin-like domain mediates DDR2 interactions with collagens I, III, and X (3-5). Collagens II and V are less efficacious ligands (3). DDR2 selectively recognizes the triple helical structure of collagen compared to monomeric or denatured collagen (3, 5, 6). Within collagen II, the D2 period is required for DDR2 binding, and the D1 period is additionally required to trigger DDR2 autophosphorylation (6). The ECD of DDR2 exists as a non-covalent dimer in solution, and dimerization of the receptor greatly enhances collagen binding (4, 7). DDR2 interaction with collagen I inhibits collagen fibrillogenesis and alters collagen fiber morphology (7). Ligand binding induces DDR2 autophosphorylation in the cytoplasmic domain (3, 5, 8), which promotes associations with Shc and Src (9). In addition to the above mechanism, DDR2 exhibits a distinct interaction with collagen X. A region other than the discoidin-like domain of DDR2 recognizes the non-helical NC1 domain of collagen X, and this interaction does not lead to receptor autophosphorylation (5). Activation of DDR2 by collagen induces upregulation of MMP-1, -2, and -13 as well as DDR2 itself (3, 8, 10). DDR2 is implicated in collagenous matrix destruction and cell invasiveness (8, 10). DDR2 is also upregulated in several pathological conditions, including hepatic fibrosis following injury, rheumatoid and osteoarthritis, and smooth muscle cell hyperplasia (8, 10-12).

1. Vogel, W.F. et al. (2006) Cell. Signal. 18:1108.
2. Karn, T. et al. (1993) Oncogene 8:3433.
3. Vogel, W. et al. (1997) Mol. Cell 1:13.
4. Leitinger, B. (2003) J. Biol. Chem. 278:16761.
5. Leitinger, B. and A.P.L Kwan (2006) Matrix Biol. 25:355.
6. Leitinger, B. et al. (2004) J. Mol. Biol. 344:993.
7. Mihai, C. et al. (2006) J. Mol. Biol. 361:864.
8. Olaso, E. et al. (2001) J. Clin. Invest. 108:1369.
9. Ikeda, K. et al. (2002) J. Biol. Chem. 277:19206.
10. Xu, L. et al. (2005) J. Biol. Chem. 280:548.
11. Wang, J. et al. (2002) J. Autoimmun. 19:161.
12. Ferri, N. et al. (2004) Am. J. Pathol. 164:1575.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for DDR2 Antibody (AF2538)(16)

Publications using AF2538

• T Han, P Mignatti, SB Abramson, M Attur Periostin interaction with discoidin domain receptor-1 (DDR1) promotes cartilage degeneration PLoS ONE, 2020-04-24;15(4):e0231501. 2020-04-24 [PMID: 32330138] (Western Blot, Human)
• AA Jalan, D Sammon, JD Hartgerink, P Brear, K Stott, SW Hamaia, EJ Hunter, DR Walker, B Leitinger, RW Farndale Chain alignment of collagen I deciphered using computationally designed heterotrimers Nat. Chem. Biol., 2020-01-06;0(0):. 2020-01-06 [PMID: 31907373] (Western Blot, Human)
• Elizabeth V. Nguyen, Brooke A. Pereira, Mitchell G. Lawrence, Xiuquan Ma, Richard J. Rebello, Howard Chan et al. Proteomic Profiling of Human Prostate Cancer-associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the Tumor Microenvironment* Molecular & Cellular Proteomics 7/1/2019 [PMID: 31061140]
• Megan M. Tu, Francis Y. F. Lee, Robert T. Jones, Abigail K. Kimball, Elizabeth Saravia, Robert F. Graziano et al. Targeting DDR2 enhances tumor response to anti–PD-1 immunotherapy Science Advances 2/20/2019 [PMID: 30801016]
• David A. Yeung, Nirvan Shanker, Anjum Sohail, Brent A. Weiss, Carolyn Wang, Jack Wellmerling et al. Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I Journal of Molecular Biology 1/1/2019 [PMID: 30458172]
• Victoria Juskaite, David S Corcoran, Birgit Leitinger Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers eLife 6/7/2017 [PMID: 28590245]
• I Majkowska, Y Shitomi, N Ito, NS Gray, Y Itoh Discoidin Domain Receptor 2 Mediates Collagen-Induced Activation of Membrane-Type 1 Matrix Metalloproteinase in Human Fibroblasts J. Biol. Chem, 2017-03-07;0(0):. 2017-03-07 [PMID: 28270508] (Western Blot, Human)
• Bo An, Vittorio Abbonante, Huifang Xu, Despoina Gavriilidou, Ayumi Yoshizumi, Dominique Bihan et al. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor* Journal of Biological Chemistry 2/26/2016 [PMID: 26702058]
• Bo An, Vittorio Abbonante, Huifang Xu, Despoina Gavriilidou, Ayumi Yoshizumi, Dominique Bihan et al. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor* Journal of Biological Chemistry 2016-02-26 [PMID: 26702058] (Western Blot, Human)
• Iwai L, Payne L, Luczynski M, Chang F, Xu H, Clinton R, Paul A, Esposito E, Gridley S, Leitinger B, Naegle K, Huang P Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants. Biochem J, 2013-09-15;454(3):501-13. 2013-09-15 [PMID: 23822953] (Western Blot, Human)
• Huifang Xu, Dominique Bihan, Francis Chang, Paul H. Huang, Richard W. Farndale, Birgit Leitinger Discoidin Domain Receptors Promote alpha 1 beta 1- and alpha 2 beta 1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation PLoS ONE 12/20/2012 [PMID: 23284937]
• DDR2 plays a role in fibroblast migration independent of adhesion ligand and collagen activated DDR2 tyrosine kinase. Biochem Biophys Res Commun, 2012-11-03;429(1):39-44. 2012-11-03 [PMID: 23131558] (ICC, Immunoprecipitation, Western Blot, Human)
• Kawai I, Hisaki T, Sugiura K, Naito K, Kano K Discoidin domain receptor 2 (DDR2) regulates proliferation of endochondral cells in mice. Biochem Biophys Res Commun, 2012-09-26;427(3):611-7. 2012-09-26 [PMID: 23022180] (Western Blot, Mouse)
• Xu H, Raynal N, Stathopoulos S, Myllyharju J, Farndale RW, Leitinger B Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1. Matrix Biol., 2010-10-28;30(1):16-26. 2010-10-28 [PMID: 21044884] (Western Blot, Human)
• Bassam R. Ali, Huifang Xu, Nadia A. Akawi, Anne John, Noushad S. Karuvantevida, Ruth Langer et al. Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients Human Molecular Genetics 6/1/2010 [PMID: 20223752]
• Bassam R. Ali, Huifang Xu, Nadia A. Akawi, Anne John, Noushad S. Karuvantevida, Ruth Langer et al. Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients Human Molecular Genetics 2010-06-01 [PMID: 20223752] (Immunocytochemistry, Human)

Reviews for DDR2 Antibody (AF2538) (3)

Average Rating: 4.7

(Based on 3 reviews)

We have 3 reviews tested in 1 species: Human.

reviewed by: Verified Customer WB Human 01/07/2015

Summary

• Application: Western Blot
• Sample Tested: See PMID 21335558
• Species: Human

reviewed by: Verified Customer IF Human 01/07/2015

Summary

• Application: Immunofluorescence
• Sample Tested: See PMID 23131558
• Species: Human

reviewed by: Verified Customer WB Human 01/07/2015

Summary

• Application: Western Blot
• Sample Tested: See PMID 21044884
• Species: Human

Bioinformatics

• Gene Symbol: DDR2
• Uniprot:
  • Q16832()