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: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet:

Article Snippet: C overnight with rabbit antibodies directed against α v β 3 (1:100, Abbiotec, Escondido, CA; Cat # 251672), α 5 β 1 (1:100, Novus Biological, Centennial, CO; Cat # nbp2-29788), α 5 (1:200, Millipore Sigma, Burlington, MA; Cat # AB1928), α v (1:200, Millipore Sigma, Burlington, MA; Cat # AB1930) or AXT107 (1:100, Asclepix, Baltimore, MD) in blocking solution.

Techniques: Recombinant, Protease Inhibitor, Electron Microscopy, Plasmid Preparation, Transgenic Assay, Software, Diagnostic Assay, Microinjection

Journal: PLoS ONE

Article Title: S . aureus alpha-toxin monomer binding and heptamer formation in host cell membranes – Do they determine sensitivity of airway epithelial cells toward the toxin?

doi: 10.1371/journal.pone.0233854

Figure Lengend Snippet: A: Representative examples of immune fluorescence assays using epifluorescence microscopy (nuclei counterstained using DAPI) that were performed on 16HBE14o-, S9 or A549 cells grown on coverslips using antibodies against ADAM10, the α5β1 integrin or against caveolin-1. Staining appearing in red represents specific labelling of the respective proteins. Scale bars: 10 μm. B: Semi-quantitative determination of primary and secondary antibody-mediated fluorescence in suspended individual cells by flow cytometry. During flow cytometry, the fluorescence of the antibody-tagged proteins per cell was measured and the respective medians of the detected peaks were used for calculating the means ± S.D. for the biological replicates (n = 4, each). Individual means were tested for significant differences using Student’s t-test or Welch’s t-test: * = p ≤ 0.05, ** = p ≤ 0.01 or *** = p ≤ 0.001.

Article Snippet: Antibodies (Ab) were obtained from these sources: Hla-Ab (S7531) from Sigma (Steinheim, Germany); ADAM10 Ecto (MAB1427-100) from R&D Systems and purchased through antikoerper-online.de (Aachen, Germany); Caveolin-1 (7C8) (sc-53564), normal mouse IgG 2b (sc-3879) from Santa Cruz Biotechnology (Heidelberg, Germany); Integrin α5β1 (M200) (NBP2-52680) from Novus Biologicals and purchased through Bio-Techne (Wiesbaden, Germany); Alexa Fluor ® 594 AffiniPure goat anti-mouse IgG (H+L) (115-585-003) from Jackson ImmunoResearch and purchased through Dianova (Hamburg, Germany); goat anti-rabbit IgG-HRP (7074s) and anti-rabbit IgG (H+L) F(ab') 2 Fragment Alexa Fluor ® 594 Conjugate (8889S) from Cell Signaling (Frankfurt am Main, Germany).

Techniques: Fluorescence, Epifluorescence Microscopy, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Galectin-1 Restricts Vascular Smooth Muscle Cell Motility Via Modulating Adhesion Force and Focal Adhesion Dynamics

doi: 10.1038/s41598-018-29843-3

Figure Lengend Snippet: Characterization of interaction of Gal-1 with α5β1 integrin and fibronectin. (A) SPR analysis of interaction between CSGal-1 and α5β1intergrin. Recombinant mouseα 5β1intergrin protein was immobilized on a CM5 chip and CSGal-1 at indicated concentrations were injected at a flow rate of 30 μl/min. Left panel, sensorgrams generated with serial concentrations of CSGAL-1. Middle panel, the curve showing the specific signals obtained after subtracting the background binding to control chip without α5β1intergrin immobilization. Right panel, sensorgrams of CSGal-1 (1600 nM) pre-incubated without (control) or with 10 mM sucrose or lactose prior to injection. (B) Human plasma fibronectin was immobilized on a CM chip and CSGAL-1 at indicated concentrations were injected at a flow rate of 30 μl/min. Left panel, sensorgrams generated with serial concentrations of CSGAL-1. Middle panel, the curve showing the specific signals obtained after subtracting the background binding to control chip without fibronectin immobilization. Right panel, sensorgrams of CSGal-1 (6400 nM) pre-incubated without (control) or with 5 mM sucrose or lactose prior to injection. (C) WT and Gal-1-KO VSMCs were reseeded on fibronectin-coated dishes for 16 h. Cells were incubated with 1 mM 3,3-dithiobis[sulfosuccinimidylpropionate] in PBS at 4 °C for 30 min, followed by addition of 50 mM Tris-HCl to quench unreacted crosslinker. Cells were then extracted with PBS containing 100 mM lactose, 0.1% SDS and 0.5% Triton X-100. After 3 PBS washes, crosslinked proteins were recovered by incubation with 50 mM dithiothreitol in PBS at 37 °C for 30 min. Both un-crosslinked and crosslinked proteins were subjected to Western blot analysis with specific antibodies targeting respective proteins. Uncropped images of immunoblots are shown in Supplementary Fig. .

Article Snippet: The 96-well polystyrene high bind microplate (#9018; Corning) was incubated with 100 μl of 10 μg/mL recombinant mouse α5β1intergrin protein (7728-A5, R&D systems) in 50 mM Na 2 CO 3 pH 9.6 at 4 °C overnight.

Techniques: Recombinant, Injection, Generated, Binding Assay, Control, Incubation, Clinical Proteomics, Western Blot