Journal: Journal of Dental Research

Article Title: Selective Role of Discoidin Domain Receptor 2 in Murine Temporomandibular Joint Development and Aging

doi: 10.1177/0022034517738190

Figure Lengend Snippet: Discoidin domain receptor 2 (DDR2) is preferentially expressed and activated in temporomandibular joint (TMJ) articular fibrocartilage. (A) Strategy for developing Ddr2 LacZ-tagged allele mice. Mice with recombinant allele knocked into the Ddr2 locus (“knockout first” allele) were developed as described in the methods and crossed with global Cre (Sox2-Cre) mice to generate a germline Ddr2 LacZ-tagged allele (Ddr2+/LacZ). (B–G) LacZ localization in mandibular condyle (B, D, F) and knee joint (C, E, G) from 2-mo-old Ddr2+/LacZ mice. Whole mount LacZ staining (B, C). Low- (D, E) and high-magnification (F, G) histologic sections; high-magnification images are of boxed areas in D, E. (H–J) Reverse transcription quantitative real-time polymerase chain reaction detection of Ddr2 (H), Col1a1 (I), and Col2a1 (J) mRNA in TMJ and knee articular cells from 2-mo-old wild-type mice (n = 6). Values were normalized to β-actin mRNA. (K–P) Immunohistochemistry: TMJ (K, M, O, P) and knee joint (L, N). Antibodies: anti-total DDR2 (K, L, O) and anti-phospho-DDR2 (Y740; M, N, P). (O, P) To determine background staining, TMJs from Ddr2slie/slie mice were stained with total (O) and phosphor-DDR2 (P) antibodies. Scale bars: 0.2 mm in panel B; 0.5 mm for panel C; 40 µm for panels D, E, K–P; 20 µm for panels F, G. Arrow (O) indicates defects in condylar morphology of Ddr2slie/slie mice.

Article Snippet: Sections were incubated with total DDR2 antibody (ab5520; Abcam) or phospho-DDR2 (Y740) antibody (MAB25382; R&D).

Techniques: Recombinant, Knock-Out, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal:

Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

doi: 10.1016/j.jmb.2008.10.060

Figure Lengend Snippet: Creation of stable cells lines expressing the recombinant protein DDR2/-KD. a) A schematic representation of V5 His-tagged full-length mouse DDR2 and of the V5 His-tagged membrane anchored, kinase deficient DDR2 (DDR2/-KD) transmembrane protein. The extracellular (ECD), transmembrane (TM), juxtamembrane (JM) and tyrosine kinase (TyrK) domains are indicated. The numbers denote the sequence of amino acids in our recombinant proteins. b) DDR2/-KD does not undergo collagen induced tyrosine phosphorylation. Following SDS-PAGE, Western blot of whole cell lysates from native (NT) or transiently transfected (DDR2/-KD) HEK293 cells before (−) and after (+) collagen stimulation, were probed using anti-phosphotyrosine (4G10) or anti-V5 antibodies. While the ~ 125kD band indicates phosphorylation of endogenously occurring DDRs, no phosphorylation signal was present for DDR2/-KD (indicated by arrow). c) Western blot indicating expression of endogenous DDR1 and DDR2 in 3T3 cells. d) Three stable MC3T3 cell lines, KD1, KD2 and KD3 were selected for our study, which show increasing levels of DDR2/-KD expression (arrow). (E) indicates empty lane.

Article Snippet: The expression of endogenous DDR1 and DDR2 was tested in non-transfected MC3T3 cells using Western blotting with anti DDR1 (sc532, Santa Cruz biotechnology, Inc) and anti DDR2 (R&D systems, Inc., Minneapolis, MN) antibodies.

Techniques: Expressing, Recombinant, Membrane, Sequencing, Phospho-proteomics, SDS Page, Western Blot, Transfection

Journal:

Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

doi: 10.1016/j.jmb.2008.10.060

Figure Lengend Snippet: DDR2/-KD is localized on the plasma membrane. a) Confocal images of HEK293 cells transiently transfected with DDR2/-KD and immunolabeled with an antibody against the DDR2 ECD; nontransfected cells show a low level of endogenous DDR2 while transfected cells show an increased level of DDR2 ECD on the membrane. b) Cell surface biotinylation of MC3T3 cells expressing DDR2/-KD construct, demonstrating surface localization of the truncated receptor. Cell samples were biotinylated (as indicated) and surface proteins were precipitated from the lysates with streptavidin-agarose beads (s lanes); the supernatant from the pull-down was recovered and probed for intracellular proteins (i lanes). Following SDS-PAGE, samples were analyzed by Western blotting with antibodies against V5 tag (DDR2/-KD) or ERK2 as indicated. DDR2/-KD is mainly present on the cell surface, while a lower molecular weight band (probably indicating the immature, non glycosylated receptor) is only found in the intracellular lane. The intracellular protein ERK2 is only present in the intracellular lane, demonstrating that biotinylation is specific to cell surface proteins. As an additional control, non-biotinylated samples, show that the streptavidin pull down is specific to biotinylated proteins.

Article Snippet: The expression of endogenous DDR1 and DDR2 was tested in non-transfected MC3T3 cells using Western blotting with anti DDR1 (sc532, Santa Cruz biotechnology, Inc) and anti DDR2 (R&D systems, Inc., Minneapolis, MN) antibodies.

Techniques: Clinical Proteomics, Membrane, Transfection, Immunolabeling, Expressing, Construct, SDS Page, Western Blot, Molecular Weight, Control

Journal:

Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

doi: 10.1016/j.jmb.2008.10.060

Figure Lengend Snippet: Overexpression of DDR2/-KD inhibits native banded structure of collagen fibers. Representative TEM micrographs show collagen fibers in the ECM. The native nontransfected cells show well formed collagen fibers with banded structure. In contrast, stable cell lines expressing DDR2/-KD were comprised of collagen fibers with weak or no banded structure; arrows indicate fibers with poor banded structure. All samples shown were cultured for two weeks.

Article Snippet: The expression of endogenous DDR1 and DDR2 was tested in non-transfected MC3T3 cells using Western blotting with anti DDR1 (sc532, Santa Cruz biotechnology, Inc) and anti DDR2 (R&D systems, Inc., Minneapolis, MN) antibodies.

Techniques: Over Expression, Stable Transfection, Expressing, Cell Culture

Journal:

Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

doi: 10.1016/j.jmb.2008.10.060

Figure Lengend Snippet: Overexpression of DDR2/-KD affects collagen fiber diameter. The collagen fiber diameters for all three stable cell lines expressing DDR2/-KD are significantly smaller than nontransfected cells. In the histogram plots, ‘d’ is the average fiber diameter obtained by measuring 100 fibers for each cell line. The TEM images and measurements shown here were performed on cells cultured for two weeks.

Article Snippet: The expression of endogenous DDR1 and DDR2 was tested in non-transfected MC3T3 cells using Western blotting with anti DDR1 (sc532, Santa Cruz biotechnology, Inc) and anti DDR2 (R&D systems, Inc., Minneapolis, MN) antibodies.

Techniques: Over Expression, Stable Transfection, Expressing, Cell Culture

Journal:

Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

doi: 10.1016/j.jmb.2008.10.060

Figure Lengend Snippet: Overexpression of DDR2/-KD affects collagen fiber diameter growth rate. The lateral growth of collagen fibers in native cells reaches a steady state value for fiber diameter within one week. Cells expressing DDR2/-KD not only have smaller fiber diameters at each time point, but also continue to grow laterally for 1 to 2 weeks of culture. Our results indicate that cells expressing DDR2/-KD reach a steady state value for lateral growth later than native cells.

Article Snippet: The expression of endogenous DDR1 and DDR2 was tested in non-transfected MC3T3 cells using Western blotting with anti DDR1 (sc532, Santa Cruz biotechnology, Inc) and anti DDR2 (R&D systems, Inc., Minneapolis, MN) antibodies.

Techniques: Over Expression, Expressing

Journal:

Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

doi: 10.1016/j.jmb.2008.10.060

Figure Lengend Snippet: Collagen fibrillogenesis is reduced in DDR2/-KD expressing cells. FITC-labeled collagen was added to cell samples and allowed to incubate for the indicated time points of 12 and 24 hours. The nontransfected samples show formation and growth of collagen fibers with time. Both stable and transiently transfected cells expressing DDR2/-KD show inhibition of collagen fibrillogenesis. Out of the stable DDR2/-KD cell lines KD1 shows a relatively higher level of fiber formation consistent with its lower expression level of DDR2/-KD.

Article Snippet: The expression of endogenous DDR1 and DDR2 was tested in non-transfected MC3T3 cells using Western blotting with anti DDR1 (sc532, Santa Cruz biotechnology, Inc) and anti DDR2 (R&D systems, Inc., Minneapolis, MN) antibodies.

Techniques: Expressing, Labeling, Transfection, Inhibition

Journal:

Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

doi: 10.1016/j.jmb.2008.10.060

Figure Lengend Snippet: Quantification of collagen content in the ECM by hydroxyproline (HP) assay reveals reduced deposition of collagen in DDR2/-KD cell lines. In this figure, HP(-AA) is the HP concentration in the adhered cell +ECM portion of the samples made without ascorbic acid. ΔHP is the difference in HP concentration between samples made with and without ascorbic acid. ΔHP is thus the amount of HP that is present as fibrillar collagen in the ECM. In the absence of ascorbic acid, all samples exhibited nearly the same amount of HP. Differences in ΔHP can be clearly observed for nontransfected cells vs. the cells expressing DDR2/-KD at 10 and 14 days of culture. Reduced collagen content was observed, consistent with the expression level of DDR2/-KD for stable vs. nontransfected cells.

Article Snippet: The expression of endogenous DDR1 and DDR2 was tested in non-transfected MC3T3 cells using Western blotting with anti DDR1 (sc532, Santa Cruz biotechnology, Inc) and anti DDR2 (R&D systems, Inc., Minneapolis, MN) antibodies.

Techniques: Concentration Assay, Expressing

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. Western blot analysis of DDR2 and Twist1 protein expression in established ovarian cancer cell lines B. A2780 or ES2 ovarian cancer cell lines were infected with lentivirus expressing the indicated shRNAi. Western blotting preformed using the indicated antibodies. C. Western blot of indicated proteins from whole cell lysates of OVCAR3 cells treated with 2ng/mL TGF-B to biochemically induce EMT for indicated times. D. ES2 ovarian cancer spheroids expressing the indicated shRNAi labeled with CMTCX dye (red) were added to a confluent monolayer of primary mesothelial cells that were labeled with CMFDA dye (green) and monitored over 7 hours. Images show representative mesothelial clearance at 0 and 7 hours. E. Quantification of experiment in D. >5 spheroids averaged per condition. Error bars denote SEM. **p<0.01, Student’s t test.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Western Blot, Expressing, Infection, Labeling

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A, B . Matrigel invasion assays. A. ES2 and B. A2780 cells were depleted of DDR2 or control (SCRM) and added to Matrigel coated Boyden chambers (8um pore size).Representative images and quantification (Means and S.D. for 3 individual chambers per group plotted as number of cells invading through to lower surface 4 per high powered field (hpf)). Repeated with statistical significance in 3 independent experiments. Unpaired t-test, **p<0.01, ***p<0.001. C. Collagen I invasion assays of ES2 shDDR2 or control (shSCRM) cells. Representative photographs taken at day 3 and day 6. D. Quantification of collagen invasion. 20 cells per well, 3 wells per condition quantified. Mean and s.d. are shown, unpaired t-test **p<0.01

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Control, Pore Size

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. ES2 or B. A2780 cells depleted of DDR2 or control (SCRM) were added to collagen I coated (+) or uncoated (−) plates for 6 hours. Western blotting with the indicated antibodies was performed on cell extract. Data representative of 3 independent experiments. C, D. Q-PCR analysis of mRNA isolated from C. ES2 or D. A2780 shRNA-depleted of DDR2 or transduced with scrambled control (SCRM). Three replicates of each gene were done for each experiment. Data are representative of three independent experiments. *p<0.05 student’s t test E. Gelatin zymography was conducted on the supernatants from ES2 cells depleted of DDR2 or control (SCRM) that had been plated on collagen I coated (+) or uncoated (−) plates for 24 hours. Representative zymogram and quantitation of 3 independent experiments shown.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Control, Western Blot, Isolation, shRNA, Transduction, Zymography, Quantitation Assay

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. ES2 or A2780 shRNAi-depleted of DDR2 or transduced with scrambled control (SCRM) were cultured for 6 hours under serum free conditions on 2mg/mL of collagen I, and cell free media was incubated with recombinant human fibronectin (FN). Extent of cleavage was measured at time intervals of 0.25, 1, and 16 hours by SDS Page followed by coomassie staining. B, C. Quantification of cleavage of intact FN by B. A2780 or C. ES2 plotted as a fraction of remaining intact FN as compared to control. Amount of remaining intact FN (~220 kDa) quantified by densitometry. Data from 3 independent experiments are plotted. D. Quantification of cell area when plated on fibronectin coated hydrogels for 3 hrs. Dots represent individual cell areas, with box plot overlay depicting the median and interquartile ranges of area spread. n>200 cells quantified for each group. E. Representative images of ES2 shSCRM or shDDR2 cells plated on fibronectin coated hydrogels.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Transduction, Control, Cell Culture, Incubation, Recombinant, SDS Page, Staining

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. A2780 cells stably expressing shRNA targeting sequence for scramble control (shSCRM) or DDR2 (shDDR2). Beta-Actin was used as a protein loading control. B–D. Representative images and quantification of Balb/c Nu mice injected IP with 7.5×106 A2780 shSCRM or shDDR2 cells. Tumor burden was assessed at 14 days post injection in the B entire peritoneal cavity C. Mesentary only D. Omentum only. N=10 mice per group. Means and s.d. Unpaired t-test, *P<0.05, **P≤0.01

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Stable Transfection, Expressing, shRNA, Sequencing, Control, Injection

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. Overall survival of advanced-stage III, IV serous ovarian cancers by Kaplan-Meier methods and log rank test. MST: Median survival time. B. Representative images of DDR2 immunohistochemical staining in i) early stage IA primary tumor ii) advanced stage IV primary tumor iii)mesenteric metastatic implants C. Classification of immunohistochemical analysis of DDR2 staining in normal (benign) ovary, human primary ovarian tumors, and metastatic implants.

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Immunohistochemical staining, Staining

Journal: Oncogene

Article Title: TWIST1 induces expression of Discoidin Domain Receptor2 (DDR2) to Promote Ovarian Cancer Metastasis

doi: 10.1038/s41388-017-0043-9

Figure Lengend Snippet: A. Western blot of patient derived primary ovarian tumor (POV) cells for expression of indicated proteins. B, C . A subset of POV cells were added to Matrigel coated Boyden chambers to assay invasion. B. Representative images of inserts and C. quantification plotted as number of cells invading through to lower surface per high powered field (hpf). Means and s.d. are shown for 3 individual chambers per group, 4 hpf per chamber. D. Proposed role of DDR2 in ovarian cancer metastasis

Article Snippet: The DDR2 primary antibody (R&D Systems, MAB2538) was used at a 1:500 dilution.

Techniques: Western Blot, Derivative Assay, Expressing

Journal: Molecular Medicine

Article Title: The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2

doi: 10.1186/s10020-021-00388-y

Figure Lengend Snippet: DDR2 as a receptor for cC1q in HT1080 epithelial cells. A C1q collagen tail peptide (C1qA peptide) binds with DDR2. Recombinant mouse DDR2-Fc chimera protein (7479-DR), DDR1-Fc (6416-DR), or control Fc (4460-MG) (20 μg per spot) were blotted onto the nitrocellulose membrane, and then detected using biotinylated C1qA peptide (200 nM). Representative image of three independent experiments. B , C Microtiter plates were immobilized with C1qA peptide or control peptide (8 μg/mL, B ), sDDR2 (2538-DR), or BSA (8 μg/mL, C ). sDDR2 (2538-DR; 0, 0.1, 0.5, 2.0 and 8 μg/mL, B) or C1qA peptide (0, 0.1, 0.5, 2.0 and 8 μg/mL, C ) were added to the plates. Plate-bound peptides were detected using anti-His antibody ( B ) or AP-conjugated streptavidin ( C ). The graph shows the mean ± SE of three independent experiments. *** p < 0.001. D Pull-down assay of 0, 1, and 5 μg sDDR2 and 10 μg C1qA peptide-coupled streptavidin beads. C1qA peptide-bound sDDR2 was detected by anti-DDR2 Ab (MAB25381). The input of C1qA peptide was visualized by dot blot using infrared 800-labeled streptavidin. E Wound healing effects of C1q and C1qA peptide. HT1080 cells were treated with or without 20 μg/mL C1q or 200 nM C1qA peptide for the indicated duration. The percentage of wound closure to the initial wound areas were calculated using Image J. The graph shows the mean ± SEM of four independent experiments. F sDDR2 inhibits the wound healing effect of C1q and C1qA peptides. sDDR2 (2 μg/mL) was co-incubated with C1q or C1qA peptide for 2 h, and the wound healing rate was determined as described in E. Data represent the mean ± SEM of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA). G C1qA peptide induces DDR2-autophosphorylation. HT1080 cells were treated with 20 μg/mL of collagen-I or C1q or C1qA peptide for 24 h. DDR2 phosphorylation was determined by immunoprecipitation with anti-DDR2 Ab and western blot with anti-phosphotyrosine (4G10) Ab. The bar graph shows the mean ± SEM of three independent experiments. * p < 0.05 (one-way ANOVA). H MMP-9 mRNA expression in HT1080 cells treated as described in 5G. Data represent the mean ± SEM of three independent experiments. ** p < 0.01 (one-way ANOVA)

Article Snippet: Cells were washed with cold DPBS, fixed with 4% paraformaldehyde for 30 min on ice, blocked with 3% BSA for 30 min, and then stained with or without mouse anti-DDR2 Ab (R&D Systems) and with or without rabbit anti-C1q Ab (Dako) at 4 °C overnight.

Techniques: Recombinant, Control, Membrane, Pull Down Assay, Dot Blot, Labeling, Incubation, Phospho-proteomics, Immunoprecipitation, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2

doi: 10.1186/s10020-021-00388-y

Figure Lengend Snippet: C1q binds to DDR2. A , B Microtiter plates were immobilized with 2 μg/mL collagen-I ( A ) and 8 μg/mL C1q ( B ). Respective concentrations of BSA were also coated as a negative control protein. Various concentrations of sDDR2 were added to the wells and ELISA was conducted to observe the binding. Blank well values were subtracted for analysis. C Microtiter plates were immobilized with 2 μg/mL each of sDDR2 and BSA, and various concentrations of C1q (0.1, 0.5, 2, and 8 μg/mL) were added to evaluate the binding of DDR2 with C1q. The graph shows the mean ± SEM of three independent experiments. *** p < 0.001 (one-way ANOVA). D Pull-down assay of 100 ng sDDR2 and 100 ng C1q were tested with Sepharose-based complete His-tag purification resin. Antibodies for western blot analyses are as indicated. E Surface plasmon analysis of C1q binding to DDR2. Various concentrations of C1q protein (0, 2.5, 5, 10, 20, and 40 μg/mL) were applied on an sDDR2-immobilized sensor chip CM5

Article Snippet: Cells were washed with cold DPBS, fixed with 4% paraformaldehyde for 30 min on ice, blocked with 3% BSA for 30 min, and then stained with or without mouse anti-DDR2 Ab (R&D Systems) and with or without rabbit anti-C1q Ab (Dako) at 4 °C overnight.

Techniques: Negative Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Pull Down Assay, Purification, Western Blot

Journal: Molecular Medicine

Article Title: The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2

doi: 10.1186/s10020-021-00388-y

Figure Lengend Snippet: Co-localization of C1q to DDR2-expressing cells. A DDR2 expression in the HT1080 cell line was determined using confocal microscopic images and flow cytometric analyses. B Confocal microscopy analysis showing co-localization of C1q with DDR2 in HT1080 cells. The cells were treated with 2 μg/mL C1q in the absence or presence of 2 μg/mL sDDR2. Confocal images of the cells show dual staining for DDR2 (red) and C1q (green). The phenomenon was attenuated with the addition of sDDR2. C Proximity ligation assay (PLA) of DDR2 and C1q. The graph shows the mean ± SEM of three independent experiments. *** p < 0.001, **** p < 0.0001 (one-way ANOVA)

Article Snippet: Cells were washed with cold DPBS, fixed with 4% paraformaldehyde for 30 min on ice, blocked with 3% BSA for 30 min, and then stained with or without mouse anti-DDR2 Ab (R&D Systems) and with or without rabbit anti-C1q Ab (Dako) at 4 °C overnight.

Techniques: Expressing, Confocal Microscopy, Staining, Proximity Ligation Assay

Journal: Molecular Medicine

Article Title: The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2

doi: 10.1186/s10020-021-00388-y

Figure Lengend Snippet: C1q-DDR2 binding promotes wound healing. A DDR2 phosphorylation was evaluated by immunoprecipitation with rabbit anti-DDR2 Ab and western blot with anti-phosphotyrosine Ab (4G10) or mouse anti-DDR2 Abs. HT1080 cells were incubated with the indicated concentration of collagen-I or C1q for 24 h. One representative image of three independent experiments. B HT1080 cells were grown to confluence in 12-well plates; a straight scratch was made and then treated with 20 μg/mL C1q for the indicated time. The wound areas were calculated and expressed as the percentage of wound closure to the initial wound areas. Values are presented as the means ± SEM of three independent experiments. C Western blot of DDR2 knockdown in HT1080 cells. D The wound healing assay was performed as described in 3B after transfection of DDR2 shRNA or scrambled control RNA (shSCR). Values are presented as the means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA)

Article Snippet: Cells were washed with cold DPBS, fixed with 4% paraformaldehyde for 30 min on ice, blocked with 3% BSA for 30 min, and then stained with or without mouse anti-DDR2 Ab (R&D Systems) and with or without rabbit anti-C1q Ab (Dako) at 4 °C overnight.

Techniques: Binding Assay, Phospho-proteomics, Immunoprecipitation, Western Blot, Incubation, Concentration Assay, Knockdown, Wound Healing Assay, Transfection, shRNA, Control

Journal: Molecular Medicine

Article Title: The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2

doi: 10.1186/s10020-021-00388-y

Figure Lengend Snippet: C1q induces MMP2 and MMP9 expression via p38 and ERK phosphorylation. A – C Effects of C1q on MMP2 and MMP9 production in HT1080 cells were detected via a gelatin zymography assay. The relative amounts of MMP2 ( B ) and MMP9 ( C ) were quantified using Image J. Collagen-I was used as a positive control. SE: short exposure, LE: long exposure. D – F HT1080 cells were treated with or without 20 μg/mL collagen-I and C1q for the various time points indicated. The expression of p-p38, p38, p-ERK1/2, ERK1/2, and ß-actin were detected using western blotting. DDR2 phosphorylation was tested after the immunoprecipitation of DDR2. The fold induction compared to untreated samples (NT, 0 h) was quantified using Image J. Data are presented as the means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA)

Article Snippet: Cells were washed with cold DPBS, fixed with 4% paraformaldehyde for 30 min on ice, blocked with 3% BSA for 30 min, and then stained with or without mouse anti-DDR2 Ab (R&D Systems) and with or without rabbit anti-C1q Ab (Dako) at 4 °C overnight.

Techniques: Expressing, Phospho-proteomics, Zymography Assay, Positive Control, Western Blot, Immunoprecipitation