Journal: Nmr in Biomedicine

Article Title: Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using α v β 3 ‐targeted iron oxide microparticles

doi: 10.1002/nbm.4948

Figure Lengend Snippet: (A) Representative MR images from mice bearing 4T1‐GFP macrometastases at Days 28–35. (Ai,ii) Tumours imaged with RGD‐MPIO; (Ai) Nonenhancing tumour at Day 28, (Aii) Gadolinium‐enhancing tumour at Day 35. (Aiii–iv) Tumours imaged with RDG‐MPIO; (Aiii) Nonenhancing tumour at Day 28, (Aiv) Gadolinium‐enhancing tumour at Day 35. Each column of images, from the left, shows T 1 ‐weighted postgadolinium images, T 2 *‐weighted pre‐MPIO MGE3D images, T 2 *‐weighted post‐MPIO MGE3D images, and overlays on T 2 *‐weighted MGE3D images showing hypointensities pre‐MPIO and post‐MPIO. For the overlays, the tumour‐bearing striatum is segmented in green, and the contralateral striatum segmented in pink. Hypointense voxels are shown in red. (B) Significantly increased RGD‐MPIO–induced hypointense voxels were evident in the tumour‐bearing striatum (white bars) compared with the contralateral striatum (black bars) at Day 35 (two‐way paired ANOVA, p < 0.05). (C) Significantly increased control RDG‐MPIO–induced hypointense voxels were also seen in the tumour‐bearing striatum (white bars) compared with the contralateral striatum (black bars) at Day 35 (two‐way paired ANOVA, p < 0.05). (D–E) Comparison of pooled data across all timepoints for (D) Nonenhancing tumours, and (E) Gadolinium‐enhancing tumours. (D) Mice with nonenhancing tumours administered RGD‐MPIO (white bars; n = 7) showed significantly increased MPIO‐induced hypointense voxels in the tumour‐bearing hemisphere (one‐way ANOVA, p < 0.005) than both the contralateral hemisphere and the mice administered control RDG‐MPIO (black bars; n = 13) in the tumour‐bearing hemisphere. (E) In mice with gadolinium‐enhancing tumours, significantly increased MPIO‐induced hypointense voxels were observed in the tumour‐bearing striatum compared with the contralateral striatum for both RGD‐MPIO (white bars; n = 10) and RDG‐MPIO (black bars; n = 3) (one‐way ANOVA, p < 0.0001). However, in mice receiving control RDG‐MPIO, the volume of MPIO‐induced hypointense voxels was also significantly greater than those administered RGD‐MPIO. Number of MPIO‐induced hypointense voxels, presented as postcontrast minus precontrast hypointense voxels for all data. Bars represent mean ± standard deviation; post‐hoc Holm–Sidak's tests. * p < 0.05, *** p < 0.001. MGE3D, multigradient echo three‐dimensional; MPIO, microparticles of iron oxide; RDG, Arg‐Asp‐Gly peptide, scrambled control; RGD, Arg‐Gly‐Asp peptide, targeting integrin α v β 3 .

Article Snippet: Subsequently, capillaries were filled with 200 ng/mL mouse integrin α v β 3 (7889‐AV‐050, R&D Systems, Abingdon, UK) and incubated at room temperature for 24 h. The remaining functional groups were quenched with 10 mM ethanolamine for 24 h at room temperature.

Techniques: Control, Comparison, Standard Deviation

Journal: Nmr in Biomedicine

Article Title: Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using α v β 3 ‐targeted iron oxide microparticles

doi: 10.1002/nbm.4948

Figure Lengend Snippet: Aggregations of iron in 4T1‐GFP tumour tissue. (A) Representative section of gadolinium‐enhancing tumour stained for Perls' Prussian blue (iron, blue) and counterstained with nuclear fast red. Black arrowheads indicate examples of single MPIO associated with the vascular endothelium, and red arrowheads indicate examples of iron aggregations. (B–C) Correlations between iron aggregations and tumour size in mice injected with either (B) RGD‐MPIO or (C) Control RDG‐MPIO. Linear regression analysis showed a positive correlation between number of aggregations and tumour size ( R 2 = 0.64, *** p < 0.001) in mice injected with RGD‐MPIO, but not control RDG‐MPIO, although a similar trend was evident; 95% confidence intervals are shown. (D–F) Consecutive sections stained for (D) Iba‐1 (macrophages and microglia, brown staining), (E) Prussian blue (iron), and (F) CD31 (blood vessels, brown staining) in a Day 35 mouse injected with RGD‐MPIO. The red arrow indicates a larger iron aggregation in a similar location to macrophage staining (D; Iba‐1), while the black arrow indicates single MPIO distant from macrophage staining, but close alignment with a blood vessel (F; CD31). (G–H) Double staining of 4T1‐GFP tumour tissue sections, from a mouse injected with RGD‐MPIO at the Day 35 timepoint, for Prussian blue (iron) and macrophages/microglia (Iba‐1, brown staining), indicate colocalisation of iron within macrophages/microglia (red arrows). Sections counterstained with nuclear fast red. (I) Double staining for Prussian Blue and CD31 revealed single MPIO (black arrow) bound to blood vessels (brown stained) in mice injected with RGD‐MPIO; representative image from Day 21 shown. (J) In the gadolinium‐enhancing 4T1‐GFP tumours, single endothelium‐bound MPIO are observed more often in mice injected with RGD‐MPIO ( n = 10) than control RDG‐MPIO ( n = 3, t ‐test, ** p < 0.01). (K) Histogram showing the cumulative frequency of the measured distance between the centre of the iron‐laden macrophages and the centre of the blood vessel lumen, indicating their close association with blood vessels. Scale bar = 25 μm in (A) and 10 μm in (D–I). MPIO, microparticles of iron oxide; RDG, Arg‐Asp‐Gly peptide, scrambled control; RGD, Arg‐Gly‐Asp peptide, targeting integrin α v β 3 .

Article Snippet: Subsequently, capillaries were filled with 200 ng/mL mouse integrin α v β 3 (7889‐AV‐050, R&D Systems, Abingdon, UK) and incubated at room temperature for 24 h. The remaining functional groups were quenched with 10 mM ethanolamine for 24 h at room temperature.

Techniques: Staining, Injection, Control, Double Staining

Journal: PLOS ONE

Article Title: An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi -Non Toxic

doi: 10.1371/journal.pone.0289183

Figure Lengend Snippet: (A) Pictural description of the α V β 3 adhesion assay methodology. (B) Fold change observed in crystal violet (CV) absorbance at a wavelength of 590 for wild type (WT), non-toxic (NT) C . novyi as well as the putative RGD-modified candidates (A and B) after exposure to the α V β 3 coated surface of the adhesion assay. * denotes a p value of < 0.05 when compared to any other cohort, including WT, NT, and modification Candidate B. (C) Average CV pixel count of entire integrin coated surface for candidates A and B as well as wild-type (WT) and non-toxic (NT) C . novyi that remain on the α V β 3 coated surface. ‡ denotes a p value of 0.05 when compared to any other cohort, including WT, NT, and modification Candidate B. Error bars represent standard deviation from the cumulative mean of three experimental replications (n = 6 each) for a total n = 18.

Article Snippet: Purified α V β 3 integrin (100μL of 10μg/mL carrier-free, human recombinant protein, R&D Systems Bio-Techne 3050-AV) solution was administered to the center of the corona-treated cover slips.

Techniques: Cell Adhesion Assay, Modification, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 5. Association of transmembrane protein 2 (TMEM2) with integrins via interactions between the extracellular domains. A and B, targeting of TMEM2 to focal adhesions (FAs) does not require the cyto- plasmic domain of TMEM2. In this experiment, mCherry-mTMEM2 (full length) and mCherry-mTMEM2/Δcyto (Δcyto) cells were analyzed for their in situ hyaluronan (HA) degradation activities. To allow specific analysis of the activity of the full-length mouse TMEM2 and its Δcyto deletion mutant, expression of endogenous human TMEM2 was silenced by siRNA treatment prior to the assay. A, in situ HA degradation assays were performed on substrate immobilized with FA-HA, as described in Experimental procedures section. Note that the pattern of in situ HA degradation is indistinguishable between mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 10 μm. B, immunostaining for vinculin in mCherry-mTMEM2 and mCherry-mTMEM2/Δcyto cells on the FA-HA substrate. Note that the sites of HA degradation colocalize with vinculin-immunoreactive puncta in both mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 2 μm. C–E, TMEM2 associates with integrins via extracellular interactions. C, cell surface–expressed TMEM2 is coimmunoprecipitated with integrin α5β1. mCherry-mTMEM2 cells were treated with the membrane-impermeable crosslinker 3’,3’-dithiobis(sulfosuccinimidyl

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: In Situ, Activity Assay, Mutagenesis, Expressing, Immunostaining, Membrane

Journal: Journal of Biological Chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 6. A model for the role of transmembrane protein 2 (TMEM2) in integrin-mediated cell adhesion and migration. Our results suggest that TMEM2-dependent degradation of hyaluronan (HA) is critical for cells to form strong cell–matrix adhesion on HA-rich extracellular matrix (ECM). A, high levels of HA in the ECM are inhibitory to the direct engagement of integrins to their ECM ligands. B, in the presence of TMEM2, HA in the ECM is locally removed, which generates a microenvironment that is permissible to the direct integrin–ECM engagement. C, the association between TMEM2 and integrins promotes the FA formation and maturation via further removal of HA in the vicinity of the integrin–ECM engagement. D, this in turn facilitates integrin clustering, integrin-mediated downstream signaling, and cellular responses. See the text for further discussion.

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: Migration

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A, B) Sensor chips containing immobilized biotinylated FK-M1 and FK-M3 were probed with mouse sITG αvβ6. Following consecutive analyte injections over 120 s, dissociation was monitored for 600 s (black). Sensorgrams were fitted with a 1:1 kinetic model (red). (C-E) FK saturation cell binding assays using cells with defined αvβ6/αvβ8 expression included FK-M1 binding to B16-mβ6 (C), FK-M3 binding to B16-mβ6 (D) and FK-M3 binding to B16-mβ8 (E). Parental B16 cells were included in order to subtract background levels when calculating equilibrium dissociation constant K D values by Scatchard plot analyses.

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Binding Assay, Expressing

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Overview of αvβ6 complexed with the 20mer A20M3 peptide. αv (light green), β6 (pink) and A20M3 (grey) chains are shown as cartoon traces. Side chains of the RGD motif are shown as sticks and spheres. (B) Superposition of αvβ6 in surface representation in complex with A20M1 (yellow carbons), A20M3 (grey carbons) and A20FMDV2 (salmon carbons). The position of manganese ions (cyan spheres) shown superimposed were derived from the human αvβ6/TGFβ1 complex (PDB ID: 5ffo). Labeled aa refer to the A20M3 sequence. (C) Superposition of the αvβ6/A20M3 complex onto the αvβ8 structure (blue carbons). Note that the A20M3 peptide is not shown here. Residue numbering refers to the Uniprot entry Q9Z0T9 (mouse integrin subunit β6) and the A20M3 peptide sequence (Arg16).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Derivative Assay, Labeling, Sequencing, Residue

Journal: Cancers

Article Title: Investigating the Interaction of Cyclic RGD Peptidomimetics with α V β 6 Integrin by Biochemical and Molecular Docking Studies

doi: 10.3390/cancers9100128

Figure Lengend Snippet: Inhibition of biotinylated fibronectin binding to α V β 6 integrin compared with inhibition of biotinylated vitronectin binding to α V β 3 .

Article Snippet: Recombinant human integrin α V β 6 receptor (R&D Systems, Minneapolis, MN, USA) was diluted to 1.0 μg/mL in coating buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MnCl 2 , 2 mM CaCl 2 , and 1 mM MgCl 2 .

Techniques: Inhibition, Binding Assay

Journal: Cancers

Article Title: Investigating the Interaction of Cyclic RGD Peptidomimetics with α V β 6 Integrin by Biochemical and Molecular Docking Studies

doi: 10.3390/cancers9100128

Figure Lengend Snippet: Docking best poses of ( a ) ligand 1a (green) and ( b ) ligand 1c (green) overlaid to the X-ray structure of the TGF-β3 undecapeptide (grey, α-helix represented as a ribbon) into integrin α V β 6 (from 4UM9.pdb). Only selected integrin residues involved in interactions with the ligand are shown and labeled in blue for α V and red for β 6 . Non-polar hydrogens are hidden for clarity, while intermolecular hydrogen bonds are shown as black dashed lines.

Article Snippet: Recombinant human integrin α V β 6 receptor (R&D Systems, Minneapolis, MN, USA) was diluted to 1.0 μg/mL in coating buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MnCl 2 , 2 mM CaCl 2 , and 1 mM MgCl 2 .

Techniques: Labeling

Journal: Cancers

Article Title: Investigating the Interaction of Cyclic RGD Peptidomimetics with α V β 6 Integrin by Biochemical and Molecular Docking Studies

doi: 10.3390/cancers9100128

Figure Lengend Snippet: Docking best poses of ( a ) ligands 2 (red), 4 (green) and 5 (blue) and ( b ) ligands 3 (red), 6 (green) and 7 (blue) into integrin α V β 6 (α V surface in grey, β 6 surface in yellow). The X-ray structure of the TGF-β3 α-helix portion is shown as a grey ribbon. Ligand aromatic rings are represented as space-filling spheres.

Article Snippet: Recombinant human integrin α V β 6 receptor (R&D Systems, Minneapolis, MN, USA) was diluted to 1.0 μg/mL in coating buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MnCl 2 , 2 mM CaCl 2 , and 1 mM MgCl 2 .

Techniques: