Journal: Journal of molecular biology

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

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 1. (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), either with 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: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

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 I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 2. (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 incu- bated with 200 μg/ml of neutralized collagen I in 96-well plates at 37 °C.

Article Snippet: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

Techniques: Binding Assay, Inhibition

Journal: Journal of molecular biology

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

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 3. 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 panels e and f and in Table 1.

Article Snippet: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

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 I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 4. 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 the addition of collagen “C” as indicated. Insets (in panels 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. Endogenous DDR expression in MC3T3-E1 cells was evaluated using Western blotting panel h. Lysates of MC3T3-E1 cells were resolved by reducing 7.5% SDS-PAGE followed by immunoblot analyses using the indicated DDR antibodies. Asterisk (*) shows a non-specific band. 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 b 0.05) and persist at 4 h. After 4 h 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 h 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 h of collagen stimulation is shown in panel j.

Article Snippet: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

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

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 6. 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 h 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: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

Techniques: 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 I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 7. 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 h 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 a indicates an additional band detected with D1G6 antibodies (discussed in the Results section). Asterisks (*) show non-specific bands.

Article Snippet: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

Techniques: Western Blot, Expressing, Phospho-proteomics, Transfection, SDS Page, Control

Journal: Journal of molecular biology

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

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Fig. 10. ICC performed on permeabilized cells for evaluating spatial distribution of receptor phosphorylation in DDR2- GFP-expressing cells after (a) 30 min and (b) 4 h 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 h), a number of filamentous structures formed in DDR2-GFP expressing cells co-localized with Y740 signal (b). The second row in panel 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: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

Techniques: Phospho-proteomics, 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 I.

doi: 10.1016/j.jmb.2018.11.015

Figure Lengend Snippet: Scheme 1. 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 h) times after collagen I stimulation. Phosphorylation of both DDR1 and DDR2 (arrow 2) co- localizes 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 h) 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 panels 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: Mouse monoclonal (MAB25381) and goat polyclonal (AF2538) anti-DDR2 antibodies (raised against a peptide sequence in the DDR2 ECD) and rabbit monoclonal against phospho-DDR2 (DDR2-Y740, catalog no. MAB25382) were obtained from R&D Systems, Minneapolis, MN.

Techniques: Phospho-proteomics, Binding Assay

Journal: Genome Biology and Evolution

Article Title: Conservation, Divergence, and Functions of Centromeric Satellite DNA Families in the Bovidae

doi: 10.1093/gbe/evz061

Figure Lengend Snippet: —SatDNA isolation and mapping onto Bos taurus (BTA), Tragelaphini, Capra hircus (CHI), and Ovis aries (OAR) chromosomes. ( a ) satDNAs amplicons obtained by PCR from the genomic DNA of the species analyzed. PCR amplicons were SATI , 400 bp; SATIV , 604 bp; SAT1.723 , 680 bp; SAT1.711a , 400 bp; and SAT1.711b , 975 bp. SATI amplicons from the Tragelaphini genomes are 500 bp long. SAT1.723 amplicons from TAN, TIM, TDE, and TOR are ∼600 bp in length, and those of the SAT1.723 amplicons from TSC, TSP, and TST are ∼750 bp. TOR and TDE SATIV sequences revealed an amplicon size of 700 bp. ( b – l ) Physical mapping of the satDNAs present at pericentromeric and centromeric regions by in situ hybridization (red or green) in the respective species chromosomes (blue, DAPI). The name and color of each probe were indicated within each metaphase. Scale bar represents 10 μm.

Article Snippet: The third tribe, the Caprini, was represented by the sheep Ovis aries (OAR) and goat Capra hircus (CHI).

Techniques: Isolation, Amplification, In Situ Hybridization

Journal: iScience

Article Title: Zanubrutinib delays selinexor resistance evolution in biopsy sample-derived primary central nervous system lymphoma models

doi: 10.1016/j.isci.2024.109799

Figure Lengend Snippet:

Article Snippet: Goat antimouse , ZSGB-BIO , ZB-2305.

Techniques: Recombinant, Protease Inhibitor, Nucleic Acid Electrophoresis, TUNEL Assay, Bicinchoninic Acid Protein Assay, Software

Journal: microPublication Biology

Article Title: The CRISPR/Cas9 system forms a condensate in the yeast nucleus

doi: 10.17912/micropub.biology.001039

Figure Lengend Snippet:

Article Snippet: anti-Mouse (HRP) , Secondary , Goat polyclonal , Promega , W4021.

Techniques: