α 5 β 1 proteins  (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: Force 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: Nature medicine

Article Title: A tripartite complex of suPAR, APOL1 risk variants and α v β 3 integrin on podocytes mediates chronic kidney disease

doi: 10.1038/nm.4362

Figure Lengend Snippet: APOL1 forms high-affinity interactions with suPAR and αvβ3 integrin. All sensorgrams were generated using SPR assays. Rate constants (ka and kd) were determined by kinetic fitting of the sensorgrams using a one-to-one binding equation, and equilibrium dissociation constant (KD) values were determined by calculating kd/ka. (a) Sensorgrams of suPAR (0–300 nM) binding to immobilized APOL1 protein variants (G0, G1 and G2), and calculated KD values. (b) Sensorgrams of αvβ3 integrin (0–10 nM) binding to immobilized suPAR in the absence (inactive form) and presence (active form) of Mn2+ (0.5 mM), and calculated KD values. (c,d) Steady-state affinity fitting curves of αvβ3 (c) or α3β1 (d) integrin (0–40 nM) binding to immobilized APOL1 (G0, G1 and G2) in the absence (inactive) and presence (active) of Mn2+ (0.5 mM). (e) Sensorgrams of αvβ3 integrin (0–20 nM) binding to immobilized APOL1 (G0, G1 and G2) in the presence of Mn2+ (0.5 mM), and calculated KD values. (f) Bar graphs showing the determined KD values for suPAR in the presence of αvβ3 (5 nM), increasing concentrations of suPAR (0–300 nM) and immobilized APOL1 proteins (G0, G1 and G2) in the absence (inactive) or presence (active) of Mn2+ (0.5 mM). The average KD values were determined from two independent experiments, and error bars for KD measurements indicate s.e.m. (n = 2) (a–f). A, analyte; IM, immobilization.

Article Snippet: Human recombinant protein integrin α v β 3 (Millipore, cc1018, R&D, 3050-AV), human integrin α 3 β 1 (R&D, 2840-A3), human HDL full-length protein (Abcam, ab77897) and human suPAR (R&D, 807-UK-100/CF) were purchased.

Techniques: Generated, Binding Assay

Journal: Nature medicine

Article Title: A tripartite complex of suPAR, APOL1 risk variants and α v β 3 integrin on podocytes mediates chronic kidney disease

doi: 10.1038/nm.4362

Figure Lengend Snippet: High levels of suPAR are needed to synergize with APOL1 G1 and G2 to induce αvβ3 integrin activation on human podocytes. (a,b) Representative western blot images for immunoprecipitation assay using HEK293T cells transfected with plasmids expressing APOL1 G0, Flag-tagged suPAR and Myc-tagged β3 integrin in different combinations, as indicated. Data are representative of at least three independent experiments. The empty pFlag-CMV3 vector was used as a negative control. (a) Cell lysates were immunoprecipitated using a mouse monoclonal anti-Flag (M2) or monoclonal anti-Myc antibody. (b) Cell lysates were immunoprecipitated using a rabbit polyclonal anti-APOL1. The cell lysates and immunoprecipitates were analyzed by immunoblotting with antibodies, as indicated. Analysis of the input (1%) indicates that the levels of APOL1, β3 integrin and suPAR are overexpressed in the transfected cells. IP, immunoprecipitation. (c) Bar graph representing level of activated β3 integrin detected in human podocytes cultured in healthy serum without (Con) or with added proteins: suPAR (2,000, 5,000 and 10,000 pg/ml), APOL1 G0, G1 and G2 (15 µg/ml) or a combination of both proteins. Data represent measurements of >50 cells and are plotted as means ± s.d. (n = 3). ***P < 0.001. Statistical significance was determined by unpaired two-tailed Student’s t-test. (d) Immunofluorescence analysis of human podocytes grown in human serum in the absence (Con) or presence of suPAR (5,000 pg/ml) and three APOL1 variant proteins (15 µg/ml). Cells were stained with AP5 antibody that specifically recognizes the active form of β3 integrin, and with anti-paxillin antibody to mark focal adhesions. Scale bar, 25 µm. (e) Bar graph representing the level of β3-integrin activation detected in human podocytes cultured in healthy serum without (Con) or with three serum samples from individuals with FSGS carrying the nonrisk APOL1 genotype, and three different serum samples from individuals with FSGS carrying two APOL1 risk-allele genotypes. FSGS-APOL1 serum #2, and to a substantial degree, serum #3, detached the cells such that the quantification of β3-integrin activation was either not determined (n.d.) or not accurate (striped bar graph). Data represent measurements of >50 cells and are plotted as means ± s.d. (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test. (f,g) Bar graph representing the level of β3-integrin activation detected in human podocytes cultured in healthy serum without (Con) or with the addition of the indicated concentrations of the proteins. In g, the concentration of suPAR was 5,000 pg/ml. Data represent measurements of >50 cells and are plotted as means ± s.d. (n = 3). *P < 0.01; ***P < 0.001. Statistical significance was determined by unpaired two-tailed Student’s t-test.

Article Snippet: Human recombinant protein integrin α v β 3 (Millipore, cc1018, R&D, 3050-AV), human integrin α 3 β 1 (R&D, 2840-A3), human HDL full-length protein (Abcam, ab77897) and human suPAR (R&D, 807-UK-100/CF) were purchased.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Negative Control, Cell Culture, Two Tailed Test, Immunofluorescence, Variant Assay, Staining, Concentration Assay

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: Pharmacology Research & Perspectives

Article Title: An integrin antagonist ( MK ‐0429) decreases proteinuria and renal fibrosis in the ZSF 1 rat diabetic nephropathy model

doi: 10.1002/prp2.354

Figure Lengend Snippet: (A) The expression of various integrins in primary human kidney cells. Following immunoprecipitation with an anti‐ α v antibody, the α v β 1, α v β 3, α v β 5, and α v β 6 heterodimers were detected by automated western analysis using antibodies that recognize the individual β ‐subunit. GAPDH level in total cell lysate was used as loading control. Human primary kidney fibroblasts ( HPKF ), human primary renal proximal tubule epithelial cells ( RPTEC ), and normal human mesangial cells ( NHMC ). IP , immunoprecipitation; IB , immunoblotting. (B) MK ‐0429 inhibits podocyte motility in Oris cell migration assay. Cells were exposed to puromycin ( PAN , 15 μ g/mL) for 24 h in the presence or absence of MK ‐0429 at different concentrations (ranging from 0.01 nmol/L to 10 μ mol/L, n = 8 for each concentration). ** P < 0.01, one‐way ANOVA . (C) MK ‐0429 suppresses the expression of fibrosis marker genes in HPKF s under basal condition or upon TGF ‐ β stimulation ( n = 8 for each group). PAI ‐1, plasminogen activator inhibitor‐1; α SMA , α ‐smooth muscle actin; Col IA 1, Collagen type I alpha‐1 chain; CTGF , connective tissue growth factor.

Article Snippet: Human recombinant α 1 β 1, α 2 β 1, α 3 β 1, α 4 β 1, α 6 β 1, α 9 β 1, α 10 β 1, α 11 β 1, and α IIb β 3 integrins from R&D Systems were reconstituted in assay buffer and Sypro orange (Sigma‐Aldrich, St. Louis, MO).

Techniques: Expressing, Immunoprecipitation, Western Blot, Control, Cell Migration Assay, Concentration Assay, Marker

Journal: Pharmacology Research & Perspectives

Article Title: An integrin antagonist ( MK ‐0429) decreases proteinuria and renal fibrosis in the ZSF 1 rat diabetic nephropathy model

doi: 10.1002/prp2.354

Figure Lengend Snippet: In vitro selectivity of MK‐0429 against various integrin subtypes

Article Snippet: Human recombinant α 1 β 1, α 2 β 1, α 3 β 1, α 4 β 1, α 6 β 1, α 9 β 1, α 10 β 1, α 11 β 1, and α IIb β 3 integrins from R&D Systems were reconstituted in assay buffer and Sypro orange (Sigma‐Aldrich, St. Louis, MO).

Techniques: In Vitro, Inhibition, 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: 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