reference identifiers additional information antibody anti human mouse y740pddr2  (R&D Systems)

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