Human DDR2 Antibody

Detection of Human DDR2 by Simple Western^TM. 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 the 12-230 kDa separation system.

Detection of Human DDR2 by Knockdown Validated 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 (https://linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human DDR2 by Western Blot 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 (https://linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human DDR2 by Knockdown Validated 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 (https://linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human DDR2 by Knockdown Validated 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 (https://linkinghub.elsevier.com/retrieve/pii/S0021925820365789), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human DDR2 Antibody by Western Blot 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 (https://pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human DDR2 Antibody by Western Blot 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 http://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 (https://pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human DDR2 Antibody by Western Blot 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 http://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 (https://pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human DDR2 Antibody by Western Blot 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 http://www.biochemj.org/bj/454/bj4540501add.htm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23822953), licensed under a CC-BY license. Not internally tested by R&D Systems.