recombinant human α 5 β 1 integrin  (R&D Systems)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Enhanced radiotheranostic targeting of integrin α 5 β 1 with PEGylation-enabled peptide multidisplay platform (PEGibody): A strategy for prolonged tumor retention with fast blood clearance

doi: 10.1016/j.apsb.2024.07.006

Figure Lengend Snippet: In vitro binding affinity and stability studies of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303. (A) SPR sensorgrams demonstrating the binding affinity of QM-2301, QM-2302, and QM-2303 for human integrin α 5 β 1 in a concentration-dependent manner. (B) The equilibrium dissociation constant ( Κ D ) of each peptide was calculated based on SPR measurements. The K D values of each precursor are shown. (C, D) Schematic diagram of the binding patterns of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302 (C), and [ 64 Cu]QM-2303 (D) to integrin α 5 β 1. Monomeric [ 64 Cu]QM-2301, and [ 64 Cu]QM-2302 bind to receptors in a single-network pattern. For [ 64 Cu]QM-2303, the PEGibody-based radiotracer, one PEGibody can bind to more than two integrin α 5 β 1 receptors, exhibiting better binding affinity. (E, F) The expression of integrin α 5 β 1 in B16F10 cells was analyzed by flow cytometry (E) and Western blotting (F). For flow cytometry assays, an anti-integrin α 5 + β 1 antibody was used. For Western blot assays, integrin α 5 (∼150 kDa) and integrin β 1 (∼138 kDa) were examined using two antibodies. (G) The stability of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 after coincubation with mouse serum within 1 h, as indicated by radio-HPLC. (H) I n vitro cell uptake of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 (750 KBq/mL) when incubated with B16F10 cells for different time period. (I) IC 50 of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 when inhibited with antibodies at different concentrations. ∗ P < 0.05, ∗∗ P < 0.01. All the quantitative experiments were performed independently at least three times (data are the mean ± SD, n = 3).

Article Snippet: Recombinant human integrin α 5 β 1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip using a standard amine coupling kit at a temperature of 25 °C.

Techniques: In Vitro, Binding Assay, Concentration Assay, Expressing, Flow Cytometry, Western Blot, Incubation

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: A schematic diagram of energy landscapes for protein unbinding. Two energy minima ( upper curve ) were observed for the fibronectin– α 5 β 1 -integrin interaction, but only one minimum ( lower curve ) was observed for fibronectin with the two PGs studied. ΔG represents the height of a barrier of width χ B . Here, (i) and (o) indicate the inner and outer barriers. Both curves have the same energy at large separations, so they are shifted for clarity. To see this figure in color, go online

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: Representative force spectroscopy data for the unbinding of SDC4 ( top ), decorin ( middle ), and α 5 β 1 -integrin ( bottom ) from a fibronectin-immobilized surface. The retraction speeds for these data are 2, 1, and 3 μ m/s, respectively. For clarity, the data for SDC4 and decorin are offset by 200 and 100 pN, respectively. To see this figure in color, go online

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Spectroscopy

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: Frequency distributions for extracted rupture forces, taken at different velocities for the unbinding of fibronectin from α 5 β 1 -integrin ( A ), SDC4 ( B ), and decorin ( C ). The number of measurements, n , from which the distributions were obtained, is shown in each panel. The fits (shown) are to either Gaussian or log-Gaussian models, and the modal averages of the rupture force for the corresponding loading rate were obtained. (These may be distinguished by noting that the fit to a log-Gaussian model passes through the origin.) Included errors correspond to the width of the 95% confidence interval for the fit. Standard error values for each fit, provided by statistical software package GraphPad, were significantly smaller than the error values (95% confidence interval width) quoted here. The log-Gaussian fit was used when the Gaussian fit was unsatisfactory, although the modal average is model independent. To see this figure in color, go online.

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Software

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: The dynamic force spectrum describing the unbinding between fibronectin and α 5 β 1 -integrin ( A ), SDC4 ( B ), and decorin ( C ). The linear increase of the average rupture force with the logarithm of the average loading rate for each pulling velocity, fitted using the Bell-Evans relation , is shown. Included errors correspond to the width of the 95% confidence interval from which average rupture forces were extracted. Two energy barriers were observed for the unbinding of fibronectin from α 5 β 1 -integrin.

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: Extracted Energetics for Each Energy Barrier Revealed in the Dynamic Spectra for the Unbinding of Fibronectin from Both the Inner, I, and Outer, o, Barriers of α 5 β 1 -Integrin, as well as Those for SDC4 and Decorin

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: ACS Omega

Article Title: Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels

doi: 10.1021/acsomega.7b01641

Figure Lengend Snippet: Half inhibition (i.e., IC 50 ) of the fluorescence of FITC-GRGDSP occurs at a ∼40-fold lower concentration for RGD than R-Glc-D, indicating greater affinity of RGD for integrin than R-Glc-D (A). Comparison of FP inhibition by R-Glc-D and RGE peptides (negative control). The average IC 50 value for R-Glc-D is 1115 ± 211 μM (mean ± standard deviation). RGE was tested once as a negative control, and the calculated IC 50 value was 2840 μM (B).

Article Snippet: Human recombinant integrin α 5 β 1 (R&D Systems) was diluted to 900 nM in the same Tris buffer.

Techniques: Inhibition, Fluorescence, Concentration Assay, Comparison, Negative Control, Standard Deviation

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Docking best poses of a) compound 7 (green) and b) compound 6 (green) in the crystal structure of the extracellular domain of α 5 β 1 integrin (α 5 subunit pink, β 1 subunit cyan, model from 3VI4.pdb). Only selected integrin residues involved in interactions with the ligand are shown. Non polar hydrogens are hidden for clarity, whereas intermolecular hydrogen bonds are shown as dashed lines.

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques:

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Docking binding modes: a) A and b) B of compound 3 (green) in the crystal structure of the extracellular domain of α 5 β 1 integrin (α 5 subunit pink, β 1 subunit cyan, model from 3VI4.pdb). Only selected integrin residues involved in interactions with the ligand are shown. Non polar hydrogens are hidden for clarity, whereas intermolecular hydrogen bonds are shown as dashed lines.

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques: Binding Assay

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Inhibition of biotinylated fibronectin binding to α 5 β 1 integrin compared with inhibition of biotinylated vitronectin binding to α v β 3 .

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques: Inhibition, Binding Assay

Journal: British Journal of Cancer

Article Title: Circulating galectins -2, -4 and -8 in cancer patients make important contributions to the increased circulation of several cytokines and chemokines that promote angiogenesis and metastasis

doi: 10.1038/bjc.2013.793

Figure Lengend Snippet: Galectin-induced cytokine secretion enhances expression of the endothelial cell surface adhesion molecules, which are responsible for galectin-mediated cancer cell-endothelial adhesion. ( A ) The presence of galectins induces expressions of cell surface adhesion molecules. Human micro-vascular lung endothelial cells were treated with control 1.5 μ g ml −1 BSA (red colour) or 1.5 μ g ml −1 galectins -2 (purple), -4 (brown), -8 (green), a combination of G-CSF (0.25 ng ml −1 ), IL-6 (0.15 ng ml −1 ), GRO α (1 ng ml −1 ), MCP-1 (1 ng ml −1 ) (black) for 24 h before the expressions of the HMVEC surface integrin α v β 1, VCAM-1, CD44 and E-selectin were analysed by flow cytometry. ( B ) The presence of neutralising antibodies against cell surface adhesion molecules inhibits galectins -2, -4 or -8-mediated cancer cell adhesion. Human micro-vascular lung endothelial cells were treated without or with 1.5 μ g ml −1 galectins -2, -4 or -8 for 24 h. The culture media were collected and used to assess ACA19 − cell adhesion to fresh HMVEC-Ls without or with addition of a combination of neutralising antibodies against integrin α v β 1 (10 μ g ml −1 ), CD44 (10 μ g ml −1 ), VCAM-1(10 μ g ml −1 ) and E-selectin (10 μ g ml −1 ). ** P <0.01, *** P <0.001 (ANOVA, Bonferroni).

Article Snippet: Recombinant human galectins -2, -4 and -8 (residual endotoxin levels <1.0 EU μ g −1 protein), and human Cytokine Protein Array, antibodies against CD44, E-selectin, VCAM-1 and integrin α v β 1 were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Flow Cytometry