Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Cancer Biology & Therapy

Article Title: Overexpression of DDR2 contributes to cell invasion and migration in head and neck squamous cell carcinoma

doi: 10.4161/cbt.28181

Figure Lengend Snippet: Figure 1. Expression levels of DDR2 transcripts and proteins in HNSCC clinical specimens. (A) Expression levels of DDR2 in 29 pairs of fresh primary HNSCC and corresponding normal clinical specimens were measured by qPCR. One of the low grade tumors’ transcripts (number 5) was used for normalization. (B) Immunohistochemical staining for DDR2 expression in 79 human HNSCC specimens (54 specimens of low grade tumors and 25 specimens of high grade tumors) and 39 cancer-associated normal (cancer-assoc. nl) specimens. Shown is a representative example of high grade HNSCC sample (right), low grade HNSCC sample (middle) and cancer-associated normal tissue sample (left). (C) Staining evaluation for the immunohistochemical staining of DDR2 expression. The staining grade is stratified as absent (−), weak (+), moderate (++), or strong (+++). Shown is a representative example of each grade. (D) Statistical analysis for immunohistochemical staining of DDR2 expression between human HNSCC specimens and cancer-associated normal specimens. (E) Statistical analysis for the relationship between the immunohistochemical staining of DDR2 expression and HNSCC characteristics. Data are presented as the mean ± SD or n (number of samples). Statistical significance was evaluated with the Student t test or χ2 test.

Article Snippet: Anti-DDR2 (MMB2538 and BAF2538) antibodies were purchased from R&D Corporation (R&D), anti-E-cadherin (ab11512, Abcam), anti-vimentin, anti-β-actin, anti-MMP-2, and anti-MMP-9 (Cell Signaling).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Biology & Therapy

Article Title: Overexpression of DDR2 contributes to cell invasion and migration in head and neck squamous cell carcinoma

doi: 10.4161/cbt.28181

Figure Lengend Snippet: Figure 2. Expression of DDR2, E-cadherin, and vimentin in HNSCC cell lines. (A and B) Tca8113, TB, SCC25, and FaDu cells were subjected to analysis of mRNA and protein expression of E-cadherin and vimentin by qPCR and western blot, respectively. Statistical significance was evaluated with the Student t test. Results display the mean ± SD. ***P < 0.001. (C) mRNA and protein expression of DDR2 in HNSCC cell lines was analyzed by reverse transcription PCR and western blot, respectively. β-actin was used as a loading control.

Article Snippet: Anti-DDR2 (MMB2538 and BAF2538) antibodies were purchased from R&D Corporation (R&D), anti-E-cadherin (ab11512, Abcam), anti-vimentin, anti-β-actin, anti-MMP-2, and anti-MMP-9 (Cell Signaling).

Techniques: Expressing, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Overexpression of DDR2 contributes to cell invasion and migration in head and neck squamous cell carcinoma

doi: 10.4161/cbt.28181

Figure Lengend Snippet: Figure 3. DDR2 overexpression has no effect on cell proliferation in HNSCC cells. (A) Tca8113 and FaDu cells (parental, EGFP-, and DDR2-transduced) were cultured in serum-free medium for 24 h. Thereafter, protein and RNA from these cells were extracted and analyzed for DDR2 expression using western blot and qPCR. (B) The cell growth curves of Tca8113 cells (parental, EGFP-, and DDR2-transduced) by MTT method. (C) Tca8113 cells (parental, EGFP-, and DDR2-transduced) treated by EdU were viewed and photographed using a fluorescence microscope. The staining positive rate was counted as positive cells/overall cells × 100%. For each group, ten random high-power fields (400×) were chosen and the cell number was counted at least three times. The histogram represents the staining positive rate of each group. (D) Tca8113 cells infected with Ad-DDR2 were similar to the controls by FAC assays. Statistical significance was evaluated with the Student t test. Results display the mean ± SD. ***P < 0.001.

Article Snippet: Anti-DDR2 (MMB2538 and BAF2538) antibodies were purchased from R&D Corporation (R&D), anti-E-cadherin (ab11512, Abcam), anti-vimentin, anti-β-actin, anti-MMP-2, and anti-MMP-9 (Cell Signaling).

Techniques: Over Expression, Cell Culture, Expressing, Western Blot, Fluorescence, Microscopy, Staining, Infection

Journal: Cancer Biology & Therapy

Article Title: Overexpression of DDR2 contributes to cell invasion and migration in head and neck squamous cell carcinoma

doi: 10.4161/cbt.28181

Figure Lengend Snippet: Figure 4. Overexpression of DDR2 enhances the invasion and migration in HNSCC cells. (A and B) Tca8113 and FaDu cells (parental, EGFP-, and DDR2-transduced) were incubated in serum-free medium with or without 2 µg/mL type I collagen (Col I) for 24 h. The histogram represents the average number of invaded and migrated cells in five random low-power fields (200×). The numbers from the control groups were used as control. Statistical significance was evaluated with the Student t test. Results display the mean ± SD. ***P < 0.001. (C) The cells in (A) were subjected to analysis of protein expression of MMP-2 and MMP-9 using western blot.

Article Snippet: Anti-DDR2 (MMB2538 and BAF2538) antibodies were purchased from R&D Corporation (R&D), anti-E-cadherin (ab11512, Abcam), anti-vimentin, anti-β-actin, anti-MMP-2, and anti-MMP-9 (Cell Signaling).

Techniques: Over Expression, Migration, Incubation, Expressing, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Overexpression of DDR2 contributes to cell invasion and migration in head and neck squamous cell carcinoma

doi: 10.4161/cbt.28181

Figure Lengend Snippet: Figure 5. Overexpression of DDR2 accelerates the process of hypoxia-induced EMT in HNSCC cells. (A) Tca8113 and FaDu cells (parental and DDR2-transduced) were cultured under normoxic or hypoxic condition for 48 h, and the morphology change of cells were photographed in low-power field (200×). (B) The cells in (A) were stained for the expression of E-cadherin (red) and vimentin (green) by immunocytofluorescence. Nuclei are labeled by DAPI (blue). Scale bar 10 µm. (C) The cells in (A) were used for immunoblot analysis of E-cadherin and vimentin expression.

Article Snippet: Anti-DDR2 (MMB2538 and BAF2538) antibodies were purchased from R&D Corporation (R&D), anti-E-cadherin (ab11512, Abcam), anti-vimentin, anti-β-actin, anti-MMP-2, and anti-MMP-9 (Cell Signaling).

Techniques: Over Expression, Cell Culture, Staining, Expressing, Labeling, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Overexpression of DDR2 contributes to cell invasion and migration in head and neck squamous cell carcinoma

doi: 10.4161/cbt.28181

Figure Lengend Snippet: Figure 6. DDR2 enhances the metastasis potential of HNSCC cells in vivo. Tca8113 cells (parental, EGFP-, and DDR2-transduced) (2 × 106/200 μL PBS) were simultaneously injected via the tail veins and subcutaneously into the hind legs of the 6-wk-old female nude mice. At day 42, mice were killed and the lungs were fixed in 4% formaldehyde for 24 h. (A) Tumor growth curve. The tumor growth was assessed every 3 d until day 42 by measuring two perpendicular diameters and calculating the volume in mm3. The typical photographs of tumors are shown. (B) The white colonies on the lung surface were enumerated macroscopically. Black arrows indicate lung metastatic tumors. Colonies were enumerated and the relative increases compared with the control groups were calculated. (C) Representative lung tissue sections from each group are shown (H&E; original magnification 100×). Black arrows indicate lung metastatic tumors. The number of lung metastatic foci in each group (n = 4) was calculated. (D and E) Immunostaining of MMP-2 and MMP-9 were performed on lung metastatic tumors (original magnification 200× and 400×). Shown are representative examples from EGFP- and DDR2-transduced groups. Statistical significance was evaluated with the Student t test. Results display the mean ± SD .***P < 0.001.

Article Snippet: Anti-DDR2 (MMB2538 and BAF2538) antibodies were purchased from R&D Corporation (R&D), anti-E-cadherin (ab11512, Abcam), anti-vimentin, anti-β-actin, anti-MMP-2, and anti-MMP-9 (Cell Signaling).

Techniques: In Vivo, Injection, Immunostaining

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: (a) Schematic diagram showing the structure of full length DDR2, human DDR2-Fc and mouse DDR2-V5-His constructs. SS: signal sequence; DS: discoidin domain; ECD: extracellular domain; IJXM: intracellular juxtamembrane region; TMD: transmembrane domain; ICD: intracellular domain; KD: kinase domain (b) Purified recombinant DDR2-V5-His and DDR2-Fc proteins (20 ng/lane) were resolved by SDS-PAGE under reducing conditions (+βME), with either 4–12% (w/v) Bis-Tris Gels (left panel) or under reducing and non-reducing (-βME, 100 ng/lane)) conditions with 10% SDS-PAGE (right panel). The separated protein was detected by immunoblotting using anti-epitope or anti-DDR2 antibodies as indicated.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Construct, Sequencing, Purification, Recombinant, SDS Page, Western Blot

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: (a) Solid phase binding of DDR2 ECD proteins to immobilized bovine-dermal collagen I as indicated. Binding was detected using antibodies against DDR2 ECD. (b) Inhibition of fibrillogenesis of bovine-dermal collagen I assessed using turbidity measurements. DDR2 ECD proteins (40 μg/ml) as indicated were incubated with 200 μg/ml of neutralized collagen I in 96-well plates at 37°C.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Binding Assay, Inhibition, Incubation

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: AFM height images of monomeric DDR2-V5-His and dimeric DDR2-Fc before and after binding to bovine-dermal collagen I as indicated (a-d). DDR2-V5-His and DDR2-Fc particles bound to collagen are indicated by black and white arrows respectively. Particle size distribution and average sizes are indicated in the accompanying histograms in (e) and (f) and in Table 1.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Binding Assay

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Topographic height (nm) of DDR2 ECD proteins from AFM images

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques:

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Live cell imaging of DDR1b-YFP- (a-d) and DDR2-GFP- (e-g) expressing MC3T3-E1 cells using wide-field fluorescence microscopy, before and after addition of collagen ‘C’ as indicated. Insets (in a-g) show selected regions which have been magnified from the corresponding images to visualize receptor assemblies. The location of these selected regions on the cell surface is indicated by dashed boxes. Western blotting of endogenous DDR expression in MC3T3-E1 cells is shown in (h). Fluorescence microscopy images (a-c) show that DDR1b-YFP exhibits a uniform distribution on the cell surface before collagen stimulation and results in cluster formation upon collagen stimulation. Quantitative analysis (i) indicates that the number of punctuate structures in cells significantly increases upon collagen stimulation (*p-value <0.05) and persist at 4 hrs. After 4 hrs of collagen stimulation, a subpopulation of DDR1b-YFP expressing cells also exhibits the presence of long, filamentous structures (d). DDR2-GFP exhibits a uniform distribution on the cell surface before collagen stimulation (e) and does not result in cluster formation upon collagen stimulation (f). However, at 4 hrs post collagen administration, filamentous structures were also observed in DDR2-GFP-expressing cells (g). The distribution of uninterrupted contour length of filamentous structures formed in DDR1b-YFP- and DDR2-GFP- expressing cells after 4 hrs of collagen stimulation is shown in (j).

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Live Cell Imaging, Expressing, Fluorescence, Microscopy, Western Blot

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Immunocytochemistry (ICC) performed on non-permeabilized cells for evaluating the association of collagen I with filamentous structures formed in (a) DDR1b-YFP- and (b) DDR2-GFP- expressing cells after 4 hrs of collagen stimulation. Total receptor is indicated in YFP channel (green) while staining with collagen antibodies is shown by TRITC (red). Co-localization YFP and TRITC is shown in yellow. Blue represents nuclear (DAPI) staining. Insets (in first row of each panel) show selected regions which have been magnified from the corresponding images. Selected regions from three different cells are shown in the second row in each panel. YFP/GFP positive filamentous structures had a very similar morphology as collagen fibrils and were observed to anchor the fibrils at cell edges. Collagen staining was intermittently present on or interspersed with YFP/GFP positive filamentous structures.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Immunocytochemistry, Expressing, Staining

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Pearson’s correlation coefficient (R) for co-localization of YFP/GFP signal with antibody (Ab) signals in ICC samples processed after 4 hrs of collagen administration

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques:

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Western blotting of DDR expression and phosphorylation in MC3T3 cells. MC3T3-E1 cells were transiently transfected with (a) DDR1b-YFP or (b) DDR2-GFP expression vectors and stimulated with 20 µg/ml collagen I (+), as described in Materials and Methods. After 4 hrs of collagen stimulation, the cells were lysed in RIPA buffer and equal protein concentrations (25 μg/lane) were resolved by reducing 7.5% SDS-PAGE followed by immunoblot analyses using the indicated antibodies to DDR1 or DDR2. β-actin was used as loading control. Number 1 in panel b indicates an additional band detected with D1G6 antibodies (discussed in the Results section). Asterisks (*) show non-specific bands.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Western Blot, Expressing, Transfection, SDS Page

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Immunocytochemistry (ICC) performed on permeabilized cells for evaluating spatial distribution of receptor phosphorylation in DDR2-GFP- expressing cells after (a) 30 min and (b) 4 hours of collagen stimulation. Total receptor is indicated in GFP channel (green) while staining with pDDR2 (Y740) antibodies is shown by TRITC (red). Co-localization GFP and TRITC is shown in yellow. Blue represents nuclear (DAPI) staining. Insets (in top row in each panel) show selected regions, which have been magnified from corresponding images. Little to no co-localization signal was detected, 30 min after collagen stimulation (a). At prolonged collagen stimulation (4 hrs), a number of filamentous structures formed in DDR2-GFP expressing cells co-localized with Y740 signal (b). The second row in (b) consists of selected regions from three different cells showing co-localization of filamentous structures with Y740 signal. A weak signal for Y740 (arrows) without a corresponding GFP signal can be observed in insets in (b, top row), which could correspond to endogenous pDDR2.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Immunocytochemistry, Expressing, Staining

Journal: Journal of molecular biology

Article Title: Clustering, Spatial Distribution and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen Type I

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Postulated model for spatial distribution and phosphorylation of DDRs upon collagen stimulation. DDR1 and DDR2 exist as homodimers on the cell surface. (a) Upon addition of monomeric collagen I, DDR1 and DDR2 interact with collagen, which leads (arrow 1) to the assembly of the receptors into filamentous structures aligned with collagen fibrils. This process occurs at prolonged (~4 hrs) times after collagen I stimulation. Phosphorylation of both DDR1 and DDR2 (arrow 2), co-localize in these structures, as determined using Y513 (pDDR1b) as well as Y792 (pDDR1) and Y740 (pDDR2) antibodies, indicated as black and green circles. (b) In another pathway, DDR1b assembles into clusters within minutes of collagen I stimulation (likely binding to non-fibrillar collagen present at early stages of fibrillogenesis) and gets endocytosed. However, phosphorylation at Y513 and Y792 is not detected in these clusters at the early (~30 min) time point. At later times (~4 hrs) post-ligand administration, phosphorylation of DDR1b in its IJXM (Y513) (green circles) but not in its KD (Y792) is detected in the DDR1b clusters. At present it is not clear if Y513-positive clusters are present on the cell-surface or constitute pools of endocytosed DDR1b receptors localizing within endosomes. Owing to the fact that DDRs contain numerous Tyr residues within the IJXM region and their KDs, phosphorylation at other Tyr residues (indicated by the red dashed circles) may also be ensuing in the structures described in (a) and (b) but are undetectable by the tools available at this time. It is likely that formation of higher-order assemblies of DDRs may recruit additional cytosolic proteins which mediate specific cellular processes.

Article Snippet: Rabbit monoclonal antibodies recognizing phosphorylated DDR1b and DDR1c (DDR1b/c-Y513), monoclonal antibody that recognize total DDR1 (D1G6), polyclonal antibody that recognize phospho-DDR1 (DDR1-Y792, catalog #11994), polyclonal antibody that recognize total DDR2 (catalog #12133) and DyLight™ 554 Phalloidin (catalog # 13054) were purchased from Cell Signaling Technology, Danvers, MA.

Techniques: Binding Assay

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, Real-time Polymerase Chain Reaction, Immunohistochemistry

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: Decreased in vitro differentiation and maturation of primary temporomandibular joint chondrocyte cultures from Ddr2slie/slie mice. Primary temporomandibular joint condyle chondrocytes were isolated from 3-mo-old wild-type (WT) and homozygous Ddr2slie/slie mice (DDR2) and grown in chondrogenesis medium for up to 15 d. Five-day cultures were used for alcian blue staining (A) and immunohistochemistry for collagen X (B). Fifteen-day cultures were stained for mineral with alizarin red (C). Cultures from day 0 to day 15 were used for reverse transcription quantitative real-time polymerase chain reaction detection of temporal changes in chondrocyte gene expression: (D) Col2a1 (Col 2), (E) cartilage oligomeric matrix protein (Comp), (F) Col10a1 (Col X), (G) matrix metalloproteinase 13 (MMP13), (H) runt-related transcription factor 2 (Runx2), and (I) bone gamma carboxyglutamate protein 2 (Bglap 2). Values were normalized to Gapdh mRNA. Scale bar: 40 µm for panels A–C.

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

Techniques: In Vitro, Isolation, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing

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: Ddr2-deficient mice exhibit tissue-selective defects in temporomandibular joint (TMJ) development. TMJ condyle structure was compared in newborn wild-type (WT) and Ddr2slie/slie mice with 3-dimensional reconstructed micro–computed tomography images (A, B), alizarin red and alcian blue whole mount staining (C, D), and safranin O staining of TMJ sections (E, F). Safranin O staining of knee joint histologic sections is also shown (G, H). Scale bars: 0.2 mm in panel A (A, B); 0.5 mm in panel C (C, D); 40 µm in panel E (E, F); 100 µm in panel G (G, H). Boxed area in panels C and D indicates alcian blue–stained region.

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

Techniques: Micro-CT, Staining

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: Spontaneous temporomandibular joint (TMJ) degeneration in 3- and 10-mo-old Ddr2slie/slie mice. (A–D) Two-dimensional micro–computed tomography (µCT) reconstruction images of TMJs from 3- and 10-mo-old wild-type (WT) and Ddr2slie/slie mice. (E–H) Three-dimensional µCT lateral views of mandibular condyles. (I–L) Three-dimensional superior views of mandibular condyles. (M–P) Safranin O staining of TMJ sections. Arrow in panel N indicates early degeneration in 3-mo-old DDR2-null section. (Q) µCT quantification of subchondral bone volume fraction at mandibular condyle surfaces (area of analysis indicated by dashed lines; see Materials and Methods). For panel Q, values are presented as ratio of bone volume / tissue volume; for panel R, as histopathologic score. (R) Histopathologic scoring of TMJ osteoarthritis based on modified Mankin score. Scale bars: 0.25 mm in panel A (A–D); 0.5 mm in panel E (E–L); 40 µm in panel M (M–P). *P < 0.05, n = 8.

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

Techniques: Micro-CT, Staining, Modification

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: Ddr2 deficiency does not affect knee joints. (A–D) Micro–computed tomography (µCT) 2-dimensional reconstruction images of knee joints from 3- and 10-mo-old wild-type (WT) and Ddr2slie/slie mice. (E–H) Three-dimensional µCT images of knee joints. (I–L) Safranin O staining of knee joint sections. (M) µCT analysis of tibia subchondral bone volume fraction (area of analysis indicated by dashed line in panel A). (N) Histopathologic scoring of knee joint osteoarthritis with modified Mankin score. Scale bars: 0.25 mm in panel A (A–D); 0.25 mm in panel E (E–H); 40 µm in panel I (I–L). *P < 0.05, n = 6.

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

Techniques: Micro-CT, Staining, Modification