Journal: International journal of molecular medicine

Article Title: Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices.

doi: 10.3892/ijmm.2013.1320

Figure Lengend Snippet: Figure 1. Phosphorylation of DDR2 in 3D collagen matrix. (A) Collagen matrices containing human fibroblasts were polymerized for 1 h (labeled 0 h) and incubated with PDGF for 4 h as shown. At the end of the incubations, cells were harvested and immunoprecipitated using phosphotyrosine antibodies (PY20), after which half of the samples were subjected to immunoblotting with antibodies (4G10) to detect phosphotyrosine protein (left panel). The major tyrosine- phosphorylated protein is indicated by an asterisk on the right. Half of the samples were subjected to silver staining and major precipitated proteins were analyzed by MALDI-TOF mass spectrometry (right panel). The results of the mass spectrometry are shown on the right. (B) RT-PCR showing that DDR2 siRNA, but not mock-transfected cells, specifically inhibited DDR2 expression. (C) Collagen matrices containing control and DDR2 silenced fibroblasts were incubated with PDGF for 4 h. At the end of the incubations, lysates were prepared and subjected to immunoblotting with antibodies (4G10) directly against the phosphotyrosine protein. The arrow indicates that the phosphotyrosine protein of 135 kDa size protein completely disappeared with DDR2 siRNA.

Article Snippet: Primary antibodies were: goat anti-human DDR2 (polyclonal) antibody from R&D Systems (Minneapolis, MN, USA) and Type I rat tail collagen (10.6 mg/ml) purchased from BD Biosciences (Bedford, MA, USA).

Techniques: Phospho-proteomics, Labeling, Incubation, Immunoprecipitation, Western Blot, Silver Staining, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Control

Journal: International journal of molecular medicine

Article Title: Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices.

doi: 10.3892/ijmm.2013.1320

Figure Lengend Snippet: Figure 2. The effect of DDR2 silencing on dendritic extensions of fibroblasts. (A) Collagen or fibronectin (FN) matrices containing mock and DDR2 siRNA- transfected fibroblasts were polymerized for 1 h with 2x104 cells/matrix. Following polymerization, samples were incubated for 4 h in DMEM containing 50 ng/ml PDGF or 10 µM LPA, as indicated. At the end of the incubations, samples were fixed and stained for actin. Selected images captured from collagen matrices are presented. Scale bar, 50 µm. (B) This is the same as in (A), except that samples were subjected to morphometric analysis. Cells were categorized as having >5 projected dendrites in the cell body as ‘+’ dendrite as shown in the representative micrographs. A total of 30-50 transfected cells were counted for each condition in ~10 fields. Results are given as percentages of cells in each category relative to the total number of cells counted.

Article Snippet: Primary antibodies were: goat anti-human DDR2 (polyclonal) antibody from R&D Systems (Minneapolis, MN, USA) and Type I rat tail collagen (10.6 mg/ml) purchased from BD Biosciences (Bedford, MA, USA).

Techniques: Transfection, Incubation, Staining

Journal: International journal of molecular medicine

Article Title: Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices.

doi: 10.3892/ijmm.2013.1320

Figure Lengend Snippet: Figure 3. The effect of DDR2 silencing on the adhesion of fibroblasts to the 3D collagen matrix. (A) Mock and DDR2-silenced cells were attached for 30 min to collagen-coated coverslips or matrices, washed and then incubated with medium containing 10 mM EDTA for 40 min. At the end of the incubations, the samples were fixed and stained for actin. Scale bar, 100 µm. (B) An enlarged view of a single cell form elsewhere in (A). DDR2 silencing caused the inhibition of small protrusions induced by EDTA around the membrane, resulting in a weak interaction of fibroblasts to the 3D collagen matrices. Scale bar, 50 µm.

Article Snippet: Primary antibodies were: goat anti-human DDR2 (polyclonal) antibody from R&D Systems (Minneapolis, MN, USA) and Type I rat tail collagen (10.6 mg/ml) purchased from BD Biosciences (Bedford, MA, USA).

Techniques: Incubation, Staining, Inhibition, Membrane

Journal: International journal of molecular medicine

Article Title: Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices.

doi: 10.3892/ijmm.2013.1320

Figure Lengend Snippet: Figure 4. The effect of DDR2 silencing on fibroblast migration in a 3D environment. (A) Floating collagen matrices were prepared containing fibroblasts previously transfected (mock or DDR2 siRNA) as indicated. Samples were incubated for 6 h in DMEM with 5 mg/ml fatty acid-free BSA and 50 ng/ml PDGF or 10 µM LPA added as shown. At the end of the incubation period, samples were fixed and the extent of the matrix contraction was measured. Results shown are averages (means ± SD) from three separate experiments, each carried out in duplicate. (B) Nested collagen matrices were prepared with precontracted floating matrices shown in (A), and incubated for 24 h in a medium containing PDGF. At the end of the incubation time, migrating cells were visualized by staining for actin (green) and propidium iodide (red). Scale bar, 100 µm. (C) Quantification of the cell migration shown in (B). Cell migration index values shown are the averages (means ± SD) of duplicate samples from three separate experiments. Compared to mock-transfected cells, DDR2-silenced fibroblasts showed decreased migration in the PDGF conditions.

Article Snippet: Primary antibodies were: goat anti-human DDR2 (polyclonal) antibody from R&D Systems (Minneapolis, MN, USA) and Type I rat tail collagen (10.6 mg/ml) purchased from BD Biosciences (Bedford, MA, USA).

Techniques: Migration, Transfection, Incubation, Staining

Journal: medRxiv

Article Title: A brain-persistent DDR2-degrading antibody reverses Alzheimer’s pathologies by restoring brain fluid dynamics and metabolic clearance

doi: 10.64898/2026.03.17.26348575

Figure Lengend Snippet: (A) CBF assessed by LSCI in WT, APP/PS1, and astrocyte specific Ddr2 overexpressing APP/PS1 mice. Left: Representative pseudocolor perfusion maps. Right: Quantification of relative perfusion units (Mean ± SEM; ** p < 0.01, one-way ANOVA). (B) Correlation between CBF (from A) and cortical Ddr2 mRNA levels (qPCR) across individual mice (Pearson r = −0.6081, p < 0.01). (C) In vivo (top) and ex vivo (bottom) whole brain fluorescence imaging after intravenous injection of the DDR2 targeting probe 1A12-mCherry. (D) Validation of 1A12-mCherry brain delivery and target specificity: Ex vivo brain sections for intrinsic mCherry fluorescence (red), anti-His tag immunofluorescence staining (green), and anti-DDR2 antibody HL2 staining (purple), the areas outlined by white squares are magnified in the adjacent panels. Scale bar: 30 μm for original images and 10 μm for enlarged images. (E) Schematic of the sequential probe injection protocol for vascular perfusion assessment: 1A12-mCherry followed 40 min later by Dextran-FITC (70 kDa). (F) Whole brain fluorescence imaging of vascular perfusion with Dextran-FITC (70 kDa). (G) Two photon microscopy of cortical vasculature. Representative images show mCherry signal (red) and dextran-FITC vasculature (green) in WT, APP/PS1, and APP/PS1-DDR2 mice, scale bars: 50 μm. (H) Representative two-photon microscopy images of vascular leakage after injection of dextran-FITC (4 kDa, green) and dextran-RB (70 kDa, red), scale bars: 50 μm. (I) Ventricular morphology analyzed by MRI based volumetric reconstruction. Left: Representative images of lateral ventricles from each group. Right: Quantification of lateral ventricular volume (Mean ± SEM; * p < 0.05, ** p < 0.01, one-way ANOVA). (J) CSF flow assessed by cisterna magna injection of high molecular weight dextran (70 kDa, green). Fluorescence stereo microscope images displaying the spatial distribution of the glymphatic system (green) in the brains ( top row ), and corresponding whole brain fluorescence imaging system scans of the same brains( bottom row ), scale bars: 2 mm.

Article Snippet: The primary antibodies used in this study were as follows: anti-DDR2 (1:1000; Servicebio, GB112568), anti-DDR2 (1:1000, R&D, AF2538), anti-BACE1 (1:50; Cell Signaling Technology, no. 5606), anti-Collagen IV (1:1000; abcam, ab6586), anti-PDGFRβ (1:1000; Cell Signaling Technology, no. 3169), anti-Occludin (1:1000; Cell Signaling Technology, no. 91131), anti-ZO1 (1:1000; abcam, ab276131), anti-β-actin (1:1000; Yeasen, 30102ES60).

Techniques: In Vivo, Ex Vivo, Fluorescence, Imaging, Injection, Biomarker Discovery, Immunofluorescence, Staining, Microscopy, High Molecular Weight