anti integrin α v β 5  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1

doi: 10.1016/j.jbc.2023.104998

Figure Lengend Snippet: Binding of CTX-Cy5 and CTX-displaying phages to various proposed target proteins and human serum albumin in the cobalt-coated bead test. “His-tag isolation and pulldown” Dynabeads were coated with various His-tagged proteins at 0.16 μM for each. The tested target proteins were MMP-2, NRP1, CLC-3 chloride channel, TIMP-2, MT1-MMP (MMP14), annexin A2 (ANX2), α v β 3 integrin (INT), MMP-9, and human serum albumin (HSA). A , the coated beads were blocked with casein and stained with 1 μM CTX-Cy5. Control beads (CONT) were not incubated with any target protein but blocked with casein and stained. B , representative intensity distribution histograms of beads from an experiment of A . C , the coated beads were blocked with casein, incubated with CTX-displaying phages (5.5 × 10 14 particles/ml), and stained to detect M13 phage binding. Control beads (CONT) were not incubated with any target protein but blocked with casein, and phages were also applied and stained. The bar graphs in A and C represent relative fluorescence intensities calculated from median fluorescence intensities of 10,000 events recorded by the flow cytometer and are presented as mean ± SD of three separate experiments with superimposed scatter plot. ∗ and ∗∗∗ indicate statistically significant difference ( p < 0.05 and p < 0.001) from control (ANOVA followed by Dunnett’s test). D , representative intensity distribution histograms of beads from the experiment of C . Note the very similar patterns of binding of fluorescent-labeled and phage-displayed CTX to various target proteins. CTX, chlorotoxin; MMP-2, matrix metalloproteinase 2; NRP1, neuropilin 1; TIMP-2, tissue inhibitor of metalloproteinase 2.

Article Snippet: The used target proteins were the following (with National Center for Biotechnology Information accession number [segment amino acid sequence]; His-tag position: C- or N-terminal; supplier; and catalog number listed in parentheses): MMP2 (NP_004521.1 [amino acids: 110–660]; N-; ProSpec, catalog no.: ENZ 769), NRP1 (NP_001019799.2 [amino acids: 2–644]; C-; Sino Biological; catalog no.: 10011-H08H); MMP-9 (NP_004985.2 [amino acids: 20–701]; C-; ProSpec; catalog no.: ENZ 1091), TIMP-2 (NP_003246.1 [amino acids: 27–220]; N-; ProSpec; catalog no.: ENZ-646), MMP 14 (NP_004986 [amino acids: 24–524]; C-; ThermoFisher; catalog no.: RP77533), ANX2 (NP_001002857.1 [amino acids: 1–339]; N-; ProSpec; catalog no.: PRO-777), α v β 3 integrin (AAA52589.1 & NP_002196.4 heterodimer; both with C-terminal His tag; Native Antigen Company; catalog no.: REC31719-100), CLC-3 (NP_001820.2 [amino acids: 1–818]; C-; Creative Biomart; custom made by recombinant expression in E. coli ), and human serum albumin (NP_000468.1 [amino acids: 25–609]; C-; Abcam; catalog no.: ab217817).

Techniques: Binding Assay, Isolation, Staining, Control, Incubation, Fluorescence, Flow Cytometry, Labeling

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: MFAP4 binding and activation of RGD-dependent integrins (A) MFAP4 promotes adhesion of human pulmonary microvascular endothelial cells (HPMECs) in a dose-dependent manner. (B) MFAP4-mediated adhesion of HPMEC can be inhibited by RGD-containing peptide (GRGDS) but not control (SDGRG) peptide. Data are means (SD) of n = 3 independent experiments. Significance is calculated by two-way ANOVA. MFI, mean fluorescence intensity. Immobilized recombinant MFAP4 (2 μg/mL) was incubated with increasing concentrations of recombinant integrins. Integrin binding to recombinant MFAP4 was detected for (C) integrins α V β 3/5/6 and αIIbβ 3 but not (D) integrins α V β 1/8 and α 4/5/8 β 1 . Used detection antibodies are shown in brackets. Data are shown as means (SD) of n = 3 independent experiments. Relative band density of (E) phosphorylated FAK (pFAK)/total FAK and (F) pERK/total ERK in HPMEC after cellular adhesion to poly-D-lysine (PDL, negative control), vitronectin (VTN, positive control), or MFAP4 are shown in representative western blots and quantitated mean (SD) of n = 3 independent experiments. Quantifications of western blotting was analyzed by two-way ANOVA for MFAP4 relative to negative control and the significance is provided for treatment factor only (independent of the significant time factor).

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Binding Assay, Activation Assay, Control, Fluorescence, Recombinant, Incubation, Negative Control, Positive Control, Western Blot

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: The AS0326 antibody blocks MFAP4s interaction with endothelial integrins (A) HPMECs were subjected to MFAP4-mediated adhesion, and inhibition of adhesion was tested with integrin α V β 3 - or integrin α V β 5 -blocking antibodies. (B) mAS0326 antibody (mAS) blocks HPMEC adhesion to MFAP4. MFI is of fluorescently labeled cells. IC, isotype control. Significance is calculated relative to the MFAP4-treated positive control in (A) and (B). Flow cytometry staining of (C) integrin α V β 3 and (D) integrin α V β 5 in HPMEC compared to IC. Representative histograms of n = 3 independent experiments are shown. Human primary retinal endothelial cells (RECs) were seeded on immobilized albumin or MFAP4. RECs were stimulated with VEGF and 24-h proliferation was assessed. (E) REC proliferation was co-treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326 (hAS). REC migration for 3.5 h on MFAP4-coated surface was assessed by Transwell assay using VEGF as chemoattractant. (F) MFAP4-dependent migration was treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326. Data are shown as individual datapoints with mean (SD) of n = 3 independent experiments. Data are normalized to albumin control. Significance is calculated relative to the hAS0326 treatment group for (E) and (F). Significance calculations are performed using one-way ANOVA followed by Dunnett’s multiple comparison test. Representative images of REC’s (purple) migrated through the pores of the Transwell assay inserts when migration on (G) albumin-coated insert or (H) MFAP4-coated insert. Bar, 100 μm. All antibodies were provided in 10 μg/mL doses for (A), (B), (E), and (F).

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Inhibition, Blocking Assay, Labeling, Control, Positive Control, Flow Cytometry, Staining, Migration, Transwell Assay, Comparison

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: Specificity of the AS0326 antibody (A) mAS0326 and (B) hAS0326 efficiently detect MFAP4 in WT ( Mfap4 +/+ ) mouse serum, but not in MFAP4-deficient ( Mfap4 −/− ) mouse serum. (C) Increasing concentrations of hAS0326-Fab or hAS0326Y94A L-CDR3 Fab variant were applied for inhibition of 1.5 nM MFAP4 binding to an excess of immobilized integrin α V β 3 . (D–F) Brown symbols, HG-HYB7-5; purple symbols, mAS0326; red symbols; hAS0326. (D) Both mAS0326 and hAS0326 efficiently detect immobilized recombinant human MFAP4 (rhMFAP4 coating) variant carrying RGD-AAA mutation (RGD-AAA), while HG-HYB7-5 antibody show RGD-dependent binding. Both mAS0326 and hAS0326 efficiently detect immobilized (E) recombinant mouse (rm) MFAP4, and (F) rhMFAP4, while HG-HYB7-5 is rhMFAP4 specific. Data are shown as means (SD) of n = 3 independent experiments.

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Variant Assay, Inhibition, Binding Assay, Recombinant, Mutagenesis

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: Structural basis for the hAS0326 Fab interaction with MFAP4 The hAS0326 Fab complex and the MFAP4 octamer. (A) Cartoon representation of the octameric MFAP4-Fab complex. (B) Each Fab contacts a single MFAP4 monomer at an epitope next to the MFAP4 N terminus but far from the binding site for the Ca 2+ ion (cyan sphere). (C) The MFAP4 octamer is built from two FIBCD1-like tetramers (colored orange and red) with eight Ca 2+ ions located at the tetramer-tetramer interface. (D) Inside view showing the concave face of the tetramer. (E) Top view of the convex face of the tetramer with the four bound Fab molecules. Orange spheres mark the first MFAP4 residue that can be located. Presumably, the disordered RGD integrin-binding motif at the N terminus of MFAP4 is trapped and inaccessible inside the funnel-shaped space formed by the Fab molecules. Nt, N terminus. (F) The epitope mapped on MFAP4. Residues primarily in contact with heavy-chain and light-chain CDRs are colored blue and green, respectively. (G) Details of the intermolecular interaction centered on MFAP4 residues 24–29. Putative polar interactions are indicated by dotted lines.

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Binding Assay, Residue

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: The macular proteome of the DL-AAA-induced model of chronic retinopathy supports integrin involvement Macular punches including choroid, RPE, and retina obtained at end-study (week 22) were analyzed by mass spectrometry and following analysis using clusterProfiler 4.0 package in R. The proteomes were generated from n = 3 eyes for non-diseased control, n = 5 eyes for DL-AAA treatment, and n = 5 eyes for hAS0326 treatment. (A) Over-representation analysis (ORA) showing the effect of DL-AAA treatment (relative to no DL-AAA treatment) and hAS0326 treatment of DL-AAA-treated eyes (relative to DL-AAA treatment), respectively. The plot was generated using compareCluster function with default settings. All ontologies with significant regulation post correction for multiple testing using the Benjamini-Hochberg procedure are shown. Dot sizes indicate the ratio (i.e., the coverage of a given term by proteins regulated for each comparison), and dot colors indicate the level of significance. (B–E) Volcano plots showing regulation of detected proteins underlying selected GO terms. For selected, significantly regulated gene ontologies, the ORA input proteins displaying significant regulation are highlighted in color. Protein IDs are shown for the top three proteins with lowest p belonging to a particular ontology. Moreover, protein IDs are shown for specific proteins of interest.

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Mass Spectrometry, Generated, Control, Comparison

Journal: Journal of Translational Medicine

Article Title: Repurposing of Zika virus live-attenuated vaccine (ZIKV-LAV) strains as oncolytic viruses targeting human glioblastoma multiforme cells

doi: 10.1186/s12967-024-04930-4

Figure Lengend Snippet: ZIKV-LAV entry in human GBM cells is mediated by Axl and integrin α v β 5 . Evaluation of the effect of siRNA-mediated knockdown of either Axl or integrin α v β 5 gene on A – B viral entry in DBTRG ( A ) and T98G ( B ) cells; C – D protein expression of Axl ( C ) and integrin α v β 5 ( D ) on the cell surface; and E – F intracellular viral replication in DBTRG ( E ) and T98G ( F ) cells. SCR , scrambled siRNA. Int.α v β 5 , integrin α v β 5. Data are presented as mean ± SEM. Non-parametric Mann–Whitney test or Kruskal–Wallis test with Dunn’s post-hoc correction was used to compare groups. p -values are shown accordingly: * p < 0.05. ** p < 0.005, *** p < 0.001

Article Snippet: The Human Protein Atlas report that Axl and integrin β 5 are highly expressed in neuroprogenitor cells (NPC) but not in terminally differentiated neurons [ , ], indicating that the GBM selectivity of ZIKV-LAV infection is partly explained by differential Axl and integrin α v β 5 expression.

Techniques: Knockdown, Expressing, MANN-WHITNEY

Journal: Journal of Translational Medicine

Article Title: Repurposing of Zika virus live-attenuated vaccine (ZIKV-LAV) strains as oncolytic viruses targeting human glioblastoma multiforme cells

doi: 10.1186/s12967-024-04930-4

Figure Lengend Snippet: Proposed model of human GBM cell death mediated by ZIKV-LAV infection. A – D General virus infection life cycle. A ZIKV-LAV enters human GBM cells through Axl and integrin α v β 5 cellular receptors. B The ZIKV-LAV genome is translated to express viral proteins and the viral genome is replicated. C The viral genome and viral proteins assemble the nucleoprotein in preparation for ( D ) release of progeny into the surroundings with concomitant viral incorporation of host cell membrane. E Cellular and viral proteins expressed during ZIKV-LAV infection are also responsible for cell death in human GBM. F – G cleavage of caspase-3 leads to non-lytic or non-inflammatory cell death by apoptosis; H – I cleavage of gasdermin-D (GSDMD) leads to the formation of membrane pore complexes that shuttles inflammatory IL-1β outside of the cells. GSDMD cleavage also leads to inflammasome activation and lytic and inflammatory cell death by pyroptosis. Image created with BioRender.com

Article Snippet: The Human Protein Atlas report that Axl and integrin β 5 are highly expressed in neuroprogenitor cells (NPC) but not in terminally differentiated neurons [ , ], indicating that the GBM selectivity of ZIKV-LAV infection is partly explained by differential Axl and integrin α v β 5 expression.

Techniques: Infection, Virus, Membrane, Activation Assay

Journal: Radiation Oncology (London, England)

Article Title: Targeting α ν β 3 and α ν β 5 inhibits photon-induced hypermigration of malignant glioma cells

doi: 10.1186/1748-717X-6-132

Figure Lengend Snippet: Photon-induced stimulation of integrin expression . A: FACS analysis of α ν β 3 (upper row) and α ν β 5 (lower row) expression with (red) and without (black) photon irradiation with single doses of 2 Gy (left) and 10 Gy (right). B: Graphical analysis of integrin α ν β 3 (gray bars) and α ν β 5 (black bars) expression with (bars) and without (red line) irradiation with single photon doses of 2, 5, and 10 Gy in U87 (upper chart) and Ln229 (lower chart) glioma cells. Display of mean value ± standard deviation (SD); statistical analysis using Student's t -test (*, indicating significance p < 0.05)

Article Snippet: Twenty-four hours after irradiation, cells were fixed with 70% ethanol and stained with a PE-labelled anibody directed against α ν β 3 (555505, BD) and a FITC-labelled antibody directed against α ν β 5 (FAB2528P, R&D).

Techniques: Expressing, Irradiation, Standard Deviation

Journal: Radiation Oncology (London, England)

Article Title: Targeting α ν β 3 and α ν β 5 inhibits photon-induced hypermigration of malignant glioma cells

doi: 10.1186/1748-717X-6-132

Figure Lengend Snippet: Inhibiton of Ln229 glioma cell migration by integrin inhibition . Quantitative analysis of Vn-based transmigration of Ln229 glioma cells following single photon doses of 0, 2, and 10 Gy without and with the addition of 50 ng/ml anti-α ν β 3 - and -α ν β 5 -antibodies and corresponding isotype controls. Display of mean value ± standard deviation (SD); statistical analysis using Student's t -test (*, indicating significance p < 0.05)

Article Snippet: Twenty-four hours after irradiation, cells were fixed with 70% ethanol and stained with a PE-labelled anibody directed against α ν β 3 (555505, BD) and a FITC-labelled antibody directed against α ν β 5 (FAB2528P, R&D).

Techniques: Migration, Inhibition, Transmigration Assay, Standard Deviation

Journal: Radiation Oncology (London, England)

Article Title: Targeting α ν β 3 and α ν β 5 inhibits photon-induced hypermigration of malignant glioma cells

doi: 10.1186/1748-717X-6-132

Figure Lengend Snippet: Inhibiton of U87 glioma cell migration by integrin inhibition . Quantitative analysis of Vn-based transmigration of U87 glioma cells following single photon doses of 0, 2, and 10 Gy without and with the addition of 50 ng/ml anti-α ν β 3 - and -α ν β 5 -antibodies and corresponding isotype controls.

Article Snippet: Twenty-four hours after irradiation, cells were fixed with 70% ethanol and stained with a PE-labelled anibody directed against α ν β 3 (555505, BD) and a FITC-labelled antibody directed against α ν β 5 (FAB2528P, R&D).

Techniques: Migration, Inhibition, Transmigration Assay