human phopho ddr1 ddr2 y796 y740 antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Investigation of cell mechanics and migration on DDR2-expressing neuroblastoma cell line

doi: 10.1101/2024.08.15.607761

Figure Lengend Snippet: A) DDR2 mRNA expression across cancer lines was analyzed in the Dependency Map Portal ( https://depmap.org/portal ; Broad Institute). B) Western blot of DDR2 expression (vs. GAPDH) upon 4 days of doxycycline treatment (0-12.5 mg/mL) on Tet-shDDR2 SHSY5Y cells. C) Cell survival/proliferation time course assay (as measured by % confluency) of Tet-shDDR2 SHSY5Y cells treated with doxycycline (on day 0) at increasing concentrations as indicated (n = 6 wells/condition). D) Western blot of DDR2 and phospho-Tyrosine 720-DDR2 (phY720-DDR2) from Tet-shDDR2 SHSY5Y cells pretreated for 3 days with the indicated concentrations of doxycycline and left unstimulated or induced with 25 mg/mL of rat tail Type I collagen for 16 hours.

Article Snippet: Primary antibodies were: from Cell Signaling Technology DDR2 (#12133); from MilliporeSigma Anti-GAPDH antibody (MAB374); and from R&D Systems human phopho-DDR1/DDR2 (Y796/Y740) antibody (MAB25382).

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: Control of craniofacial development by the collagen receptor, discoidin domain receptor 2

doi: 10.7554/elife.77257

Figure Lengend Snippet: Figure 1. Ddr2 deficiency results in impaired anterior-posterior skull growth with abnormal frontal bone and suture formation. WT and Ddr2slielsile mice were compared at 3 months (a–g) and 2 weeks (h,i). (a-c) Short snout, and reduced skull length in Ddr2slielsile mice. (a) Side (upper) and top (lower) head views of 3-month-old Ddr2slie/slie mice and WT littermates. Scale bar: 1 cm. (b) 3D rendering of μCT scans of 3-month-old skulls. Scale bar: 1 mm. (c) Linear measurements along anteroposterior axis of skulls, where SL: skull length; NB: nasal bone; CV: calvaria vault; ACB: anterior cranial base; PCB: posterior cranial base. (d) Quantification of anterior (ant.) and posterior (post.) skull width showed a selective increase only in the anterior skull of Ddr2slie/slie vs WT mice. (e) No changes were observed in skull height at any of the regions measured (anterior cranial height, ACH; middle cranial height, MCH; posterior cranial height, PCH). (f,g) μCT scans of calvarial bones and quantification showing a significant reduction of frontal bone (blue) in 3-month-old Ddr2slie/slie mice in the absence of changes in parietal (orange) or occipital (green) calvarial bones. Note frontal suture defect in Ddr2slie/

Article Snippet: After blocking, the sections were incubated at 4 °C overnight, with the following primary antibodies: anti- DDR2 (LS B15752, 1:200), anti- Y740- P- DDR2 (R&D Systems MAB25382, 1:200), anti- cleaved caspase 3 (Cell Signaling 9661, 1:200), anti- COL2 (Abcam ab34712, 1:100); anti- COL10 (Abcam ab58632, 1:100); Anti- COL1(Millipore Sigma AB765P, 1:100); Anti- GM130 (BD Biosciences 610822,1:100); anti- IBSP (1:100).

Techniques:

Journal: eLife

Article Title: Control of craniofacial development by the collagen receptor, discoidin domain receptor 2

doi: 10.7554/elife.77257

Figure Lengend Snippet: Figure 2. Cranial base hypoplasia due to chondrocyte disorganization and reduced chondrocyte proliferation in Ddr2-knockout synchondroses. (a) H&E staining (upper) and μCT scans (lower) of WT and Ddr2slie/slie skulls showing wide cranial base synchondroses. Scale bar: 500 μm. Boxed region (red) is shown in higher magnification. Br: Brain; t: Tongue. ISS: Intersphenoid synchondrosis; SOS: Spheno-occipital synchondrosis; PS: Presphenoid bone; BS: Basisphenoid bone; BO: Basis-occipital bone. In μCT scan of skulls (lower), arrowheads point to cranial base synchondroses; yellow and red lines highlight the width and height of synchondroses; respectively (Quantification is shown in b). Cyan lines highlight shortening of basisphenoid bone between ISS and SOS in Ddr2slie/slie vs WT mice (Quantification of cranial base bone lengths is shown in c); Data are presented as mean ± SD. (n=10). *p<0.01, ****p<0.0001, ns, not significant, two-tailed unpaired t test. (d), H&E staining of ISS and SOS sections showing loss of columnar organization of

Article Snippet: After blocking, the sections were incubated at 4 °C overnight, with the following primary antibodies: anti- DDR2 (LS B15752, 1:200), anti- Y740- P- DDR2 (R&D Systems MAB25382, 1:200), anti- cleaved caspase 3 (Cell Signaling 9661, 1:200), anti- COL2 (Abcam ab34712, 1:100); anti- COL10 (Abcam ab58632, 1:100); Anti- COL1(Millipore Sigma AB765P, 1:100); Anti- GM130 (BD Biosciences 610822,1:100); anti- IBSP (1:100).

Techniques: Knock-Out, Staining, Two Tailed Test

Journal: eLife

Article Title: Control of craniofacial development by the collagen receptor, discoidin domain receptor 2

doi: 10.7554/elife.77257

Figure Lengend Snippet: Figure 3. ECM defects in Ddr2-deficient synchondroses. (a-d) Immunofluorescent staining of ISS and SOS sections from 2-week-old WT and Ddr2slie/

Article Snippet: After blocking, the sections were incubated at 4 °C overnight, with the following primary antibodies: anti- DDR2 (LS B15752, 1:200), anti- Y740- P- DDR2 (R&D Systems MAB25382, 1:200), anti- cleaved caspase 3 (Cell Signaling 9661, 1:200), anti- COL2 (Abcam ab34712, 1:100); anti- COL10 (Abcam ab58632, 1:100); Anti- COL1(Millipore Sigma AB765P, 1:100); Anti- GM130 (BD Biosciences 610822,1:100); anti- IBSP (1:100).

Techniques: Staining

Journal: eLife

Article Title: Control of craniofacial development by the collagen receptor, discoidin domain receptor 2

doi: 10.7554/elife.77257

Figure Lengend Snippet: Figure 4. Ddr2 expression in craniofacial skeleton. (a) Whole-mount X-gal staining (green) of Ddr2Lacz/+ skulls showing of Ddr2 expression in midfacial region, cranial vault, and cranial sutures. Scale bar: 50 μm. (b) X-gal staining of cryostat sections of calvaria from newborn mice showing expression in suture mesenchyme, periosteum, and dura mater of flanking bones. Scale bar: 100 μm, left and 50 μm, right. (c) X-gal staining of cryostat section of ISS (top) and SOS (bottom) from newborn mice revealing Ddr2 expression in resting and proliferative chondrocyte zones, but low or undetected in terminal hypertrophic chondrocytes. Boxed regions are shown in higher magnification, right. Scale bar: 50 μm.

Article Snippet: After blocking, the sections were incubated at 4 °C overnight, with the following primary antibodies: anti- DDR2 (LS B15752, 1:200), anti- Y740- P- DDR2 (R&D Systems MAB25382, 1:200), anti- cleaved caspase 3 (Cell Signaling 9661, 1:200), anti- COL2 (Abcam ab34712, 1:100); anti- COL10 (Abcam ab58632, 1:100); Anti- COL1(Millipore Sigma AB765P, 1:100); Anti- GM130 (BD Biosciences 610822,1:100); anti- IBSP (1:100).

Techniques: Expressing, Staining

Journal: eLife

Article Title: Control of craniofacial development by the collagen receptor, discoidin domain receptor 2

doi: 10.7554/elife.77257

Figure Lengend Snippet: Figure 6. Loss of Ddr2 in Gli1-expressing cells resulted in a craniofacial phenotype similar to Ddr2slie/slie mice. (a) Protocol used for induction of Cre- recombination upon tamoxifen injection. (b) Genotyping PCR showing WT (lower band) and Ddr2 floxed alleles (upper band) near 1650 bp and recombined knockout allele below the 650 bp marker (KO). (c) Top head view showing Gli1CreERT; Ddr2fl/fl mice have short snout compared with Ddr2fl/fl mice. Scale bar: 1 cm. (d) μCT scans of Ddr2fl/fl and Gli1CreERT; Ddr2fl/fl skulls show reduced anterior-posterior skull length and increased anterior skull width (quantification in e–g). Note thinning and suture defect in the frontal bone in Gli1CreERT; Ddr2fl/fl skulls (d, bottom). Scale bar: 1 mm. (h) Quantification of frontal, parietal, and occipital bone thickness. (i) Alcian blue and Alizarin red whole mount staining shows Gli1-Cre; Ddr2fl/fl skulls have wide cranial base synchondroses compared with Gli1CreERT and Ddr2fl/fl. Scale bar: 500 μm. (j) H&E staining of ISS shows widening of resting zone and chondrocyte disorganization in Gli1CreERT; Ddr2fl/fl mice. Scale bar: 50 μm. RZ: Resting zone; PZ: Proliferative zone; HZ: Hypertrophic zone. Red bar compares RZ width. (k) Immunofluorescence images show reduced pDDR2 (red) immunostaining indicative of reduced DDR2 signaling in Gli1CreERT; Ddr2fl/fl synchondrosis. Dotted lines denote chondro-osseous junction. Boxed region is shown in higher magnification, lower panel. Cell nuclei were stained with DAPI (blue). Arrows, resting zone; arrowheads, proliferative zone. Scale bar: 50 μm. (l), quantification of immunostaining in k. (m-p) μCT images and quantification show enlarged synchondroses associated with shortening in cranial base bone lengths in 3-month-old Gli1CreERT; Ddr2fl/fl skulls compared with controls. Scale bar: 500 μm. c-h, (m-p) 3-month-old mice. (i–l) 2-week-old mice. Data are presented as mean ± SD. (n=10). *p<0.01, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, two-tailed unpaired t test.

Article Snippet: After blocking, the sections were incubated at 4 °C overnight, with the following primary antibodies: anti- DDR2 (LS B15752, 1:200), anti- Y740- P- DDR2 (R&D Systems MAB25382, 1:200), anti- cleaved caspase 3 (Cell Signaling 9661, 1:200), anti- COL2 (Abcam ab34712, 1:100); anti- COL10 (Abcam ab58632, 1:100); Anti- COL1(Millipore Sigma AB765P, 1:100); Anti- GM130 (BD Biosciences 610822,1:100); anti- IBSP (1:100).

Techniques: Expressing, Injection, Knock-Out, Marker, Staining, Immunofluorescence, Immunostaining, Two Tailed Test

Journal: eLife

Article Title: Control of craniofacial development by the collagen receptor, discoidin domain receptor 2

doi: 10.7554/elife.77257

Figure Lengend Snippet: Figure 7. Ddr2 conditional knockout in Col2a1-expressing chondrocytes causes cranial base hypoplasia and alters synchondroses without affecting cranial sutures. (a-d) μCT scans of Ddr2fl/fl and Col2a1Cre;Ddr2fl/fl skulls (3-month-old) showing reduced anterior-posterior skull length, length of individual bones and increased anterior and, to a lesser extent, posterior skull width in conditional knockout mice. Note thinning of frontal bone in Col2a1Cre;Ddr2fl/

Article Snippet: After blocking, the sections were incubated at 4 °C overnight, with the following primary antibodies: anti- DDR2 (LS B15752, 1:200), anti- Y740- P- DDR2 (R&D Systems MAB25382, 1:200), anti- cleaved caspase 3 (Cell Signaling 9661, 1:200), anti- COL2 (Abcam ab34712, 1:100); anti- COL10 (Abcam ab58632, 1:100); Anti- COL1(Millipore Sigma AB765P, 1:100); Anti- GM130 (BD Biosciences 610822,1:100); anti- IBSP (1:100).

Techniques: Knock-Out, Expressing