Journal: PLoS ONE

Article Title: Confocal Laser Scanning Microscopy Evaluation of an Acellular Dermis Tissue Transplant (Epiflex®)

doi: 10.1371/journal.pone.0045991

Figure Lengend Snippet: Summary of immunostaining with human antibodies against matrix components of the ADM; + means detectable by immunostaining, - means absence of any detectable signal.

Article Snippet: The following primary antibodies were used, each diluted 1∶100: rabbit anti-human collagen I (Rockland, USA), rabbit anti-human collagen II (Rockland, USA), rabbit anti-human collagen III (Abcam, UK), rabbit anti-human collagen IV (Rockland, USA), rabbit anti-human fibronectin (Abcam, UK), sheep anti-human hyaluronic acid (Biotrend, Germany), mouse anti-human laminin-5 (BD Bioscience, USA), rabbit anti-human laminin (Rockland, USA), mouse anti-human osteopontin (Santa Cruz Biotechnology, USA), mouse anti-human tenascin (NeoMarkers, USA), rabbit anti-human vitronectin (Biotrend, Germany), mouse anti-human thrombospondin-1 (Dianova, Germany).

Techniques: Immunostaining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Rickettsia conorii Adr1 Interacts with the C-Terminus of Human Vitronectin in a Salt-Sensitive Manner

doi: 10.3389/fcimb.2017.00061

Figure Lengend Snippet: Functional domains in vitronectin and peptides constructed for analysis of Adr1 binding region. (A) Full length vitronectin with noted functional domains is depicted in the top black arrow with the constructed peptides depicted in gray arrows below. (B) Silverstain of vitronectin peptides utilized. The first 8 peptides begin at amino acid 80 and are progressively truncated at the C-terminus. The 3 full length peptides contain a deletion within the C-terminal region. (C) E. coli expressing Adr1 binds to the 3 longest vitronectin peptides and the full length peptide with a deletion at amino acids 352–362. Equal loading is demonstrated with anti- E. coli RNA polymerase and verification of expression of Adr1 is verified using anti-Adr1. Data are representative of at least 3 replicates.

Article Snippet: Rabbit IgG anti-human vitronectin pAb was purchased from Complement Technology and sheep anti-human vitronectin IgG was from AbD Serotec.

Techniques: Functional Assay, Construct, Binding Assay, Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Rickettsia conorii Adr1 Interacts with the C-Terminus of Human Vitronectin in a Salt-Sensitive Manner

doi: 10.3389/fcimb.2017.00061

Figure Lengend Snippet: Serum resistance or sensitivity does not correlate with the ability to bind vitronectin in serum. (A,B) Western immunoblot analysis of vitronectin in serum binding to E. coli expressing loop 3 or loop 4 Adr1 mutants with both serum resistant and serum-sensitive phenotypes. Equal loading was verified using anti- E. coli RNA polymerase and Adr1 expression was verified using anti-Adr1. Data are representative of at least 3 replicates. Arrows depict Adr1 constructs that confer resistance to serum killing.

Article Snippet: Rabbit IgG anti-human vitronectin pAb was purchased from Complement Technology and sheep anti-human vitronectin IgG was from AbD Serotec.

Techniques: Western Blot, Binding Assay, Expressing, Construct

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Rickettsia conorii Adr1 Interacts with the C-Terminus of Human Vitronectin in a Salt-Sensitive Manner

doi: 10.3389/fcimb.2017.00061

Figure Lengend Snippet: Analysis of monomeric and multimeric vitronectin binding to loop 3 and 4 mutants. (A,C) Western immunoblot analysis of multimeric vitronectin binding to E. coli expressing loop 3 or loop 4 Adr1 mutants with both serum resistant and serum-sensitive phenotypes. (B,D) Western immunoblot analysis of monomeric vitronectin binding to E. coli expressing loop 3 or loop 4 Adr1 mutants with both serum resistant and serum-sensitive phenotypes. Equal loading was verified using anti- E. coli RNA polymerase and Adr1 expression was verified using anti-Adr1. Data are representative of at least 3 replicates. Arrows depict Adr1 constructs that confer resistance to serum killing.

Article Snippet: Rabbit IgG anti-human vitronectin pAb was purchased from Complement Technology and sheep anti-human vitronectin IgG was from AbD Serotec.

Techniques: Binding Assay, Western Blot, Expressing, Construct

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , The relevance of surface bound vitronectin was analyzed by challenging intact bacteria with complement active human serum in which vitronectin was depleted (HSΔ Vn ). P . aeruginosa strain SG137 was incubated in HSΔ Vn diluted in GVB++ buffer. After incubation, the cells were plated on NB agar plates and the number (CFU) of surviving bacteria was determined. B , The relevance of clusterin was analyzed by challenging intact bacteria with complement active human serum in which clusterin activity was blocked. Bacteria were incubated with anti-clusterin mAb or mouse IgG and were thereafter challenged with NHS (1%) diluted in GVB++ buffer. Incubation of bacteria in 1% HiNHS was used as a negative control. Number of bacteria (CFU) at the initiation of all experiments was defined as 100%. The mean values from three independent experiments are shown with error bars indicating SD. *, p ≤ 0.05;**, p ≤ 0.01.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: Incubation, Activity Assay, Negative Control

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , Binding of vitronectin to P . aeruginosa strain SG137 was assayed by flow cytometry. Bacteria were incubated with vitronectin (10–50 μg/ml) and bound vitronectin was detected with polyclonal vitronectin antiserum and Alexa488-labeled rabbit antiserum. Bacteria incubated with vitronectin specific antiserum and Alexa488-labeled rabbit antiserum served as controls. B , Vitronectin binds to P . aeruginosa and binding was dose-dependent. Four laboratory strains of P . aeruginosa were analyzed for vitronectin binding using a whole cell ELISA. Whole bacteria were immobilized onto microtiter plates and vitronectin (1–5 μg/ml) was added. Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit. C , Vitronectin bind to P . aeruginosa . P . aeruginosa strains SG137, ATCC 27853, NCTC 10662 and PAO1 were incubated with NHS. Bacteria were washed, lysed, separated by SDS-PAGE and analysed by Western blotting. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit. A representative experiment of three is shown. D , Vitronectin bound to both laboratory and clinical P . aeruginosa strains. Binding of vitronectin (5 μg/ml) to immobilized bacteria (0.5x10 7 ) was assayed by ELISA. Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit pAb. The mean values of three independent experiments and standard deviations (SD) are presented. Statistical significance of differences was estimated using Student’s t test. **, p ≤ 0.01; ***, p ≤ 0.001.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: Binding Assay, Flow Cytometry, Incubation, Labeling, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , Binding of clusterin to P . aeruginosa strain SG137 was assayed by flow cytometry. Bacteria were incubated with clusterin (5–25 μg/ml) and bound clusterin was detected with a monoclonal clusterin antibody and Alexa488-labeled mouse antiserum. Bacteria incubated with a monoclonal clusterin antibody and Alexa488-labeled mouse antiserum served as controls. B , Clusterin binds to intact P . aeruginosa and binding was dose-dependent. Four laboratory strains of P . aeruginosa were analyzed for clusterin binding using a whole cell ELISA. Whole bacteria were immobilized onto microtiter plates and clusterin (1–5 μg/ml) was added. Bound clusterin was detected with a monoclonal clusterin antibody followed by HRP-conjugated anti-mouse. C , Clusterin bind to P . aeruginosa . P . aeruginosa strains SG137, ATCC 27853, NCTC 10662 and PAO1 when incubated with NHS. Bacteria were washed, lysed, separated by SDS-PAGE and analysed by Western blotting. Bound clusterin was detected with a monoclonal clusterin antibody and HRP-conjugated anti-rabbit. A representative experiment of three is shown. D , Clusterin bound to both laboratory and clinical P . aeruginosa strains. Binding of clusterin (2.5 μg/ml) to immobilized bacteria (0.5x10 7 ) was assayed by ELISA. Bound clusterin was detected with a monoclonal clusterin antibody followed by HRP-conjugated anti-rabbit pAb. E , Binding of vitronectin and clusterin from NHS to immobilized P . aeruginosa were tested by whole cell ELISA. Bound vitronectin or clusterin was detected with the polyclonal vitronectin antiserum or monoclonal clusterin antibody followed by HRP-conjugated anti-rabbit or mouse pAb. The mean values of three independent experiments and standard deviations (SD) are presented. Statistical significance of differences was estimated using Student’s t test. *, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: Binding Assay, Flow Cytometry, Incubation, Labeling, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A, Localization of the region within vitronectin that mediates binding to intact P . aeruginosa . Vitronectin uses one contact region i.e. aa 354–363 to contact P . aeruginosa . Vitronectin 80–396 (Vn 80-396 ) and seven deletion mutants were expressed in HEK cells and purified. The numbers refer to amino acids residues that are included in each construct ( left panel ). Black indicates the heparin binding regions of vitronectin ( left panel ). B, Binding of serum purified full-length vitronectin and vitronectin deletion mutants (5 μg/ml) to immobilized bacteria was assayed by ELISA ( right panel ). Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit. C , Heparin inhibits binding of vitronectin to P . aeruginosa strain SG137 and to Lpd, the effect was dose-dependent. The effect of heparin (0.01–5 mg/ml) on vitronectin binding to immobilized P . aeruginosa strain SG137 was assayed. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit pAb. The mean values of three independent experiments and SD are presented. Statistical significance of differences was estimated using Student’s t test. **, p ≤ 0.01; ***, p ≤ 0.001.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: Binding Assay, Purification, Construct, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , Lpd and BSA were separated by SDS-PAGE and the two proteins were identified by silver staining. The position of the marker proteins is presented on the left. B, Vitronectin binds to Lpd. Lpd and BSA were transferred to a membrane and the membrane was incubated with vitronectin (20 μg/ml). Bound vitronectin was detected with vitronectin specific antiserum and HRP-conjugated anti-rabbit. BSA was used as a negative control. C , Vitronectin bound to immobilized Lpd and binding was dose-dependent. Binding of vitronectin (0.01–12.5 μg/ml) to immobilized Lpd was assayed by ELISA. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit pAb. D , Vitronectin binds to Lpd with an affinity of 600 ± 50 nM. Binding of Lpd or BSA used at various concentrations (0.003–50 μM) to NT-647-labeled vitronectin (50 nM) was evaluated in fluid phase by microscale themophoresis (MST). Thermophoresis was recorded at 50% LED power and 80% MST power for 30 s in a Monolith NT.115 instrument. The relative fluorescence in the thermophoresis phase of the experiment was plotted against the concentration of Lpd. E , Clusterin binds to Lpd. Lpd and BSA were separated by SDS-PAGE, transferred to a membrane and incubated with vitronectin (20 μg/ml) Bound clusterin was detected with a monoclonal clusterin antibody and HRP-conjugated anti-mouse. BSA was used as a negative control. F , Clusterin bound to immobilized Lpd and binding was dose-dependent. Binding of clusterin (0.01–10 μg/ml) to immobilized Lpd was assayed by ELISA. Bound clusterin was detected with anti-clusterin mAb and HRP-conjugated anti-mouse pAb. G , Clusterin binds to Lpd with an affinity of 654 ± 54 nM. Binding of Lpd or BSA used at various concentrations (0.001–50 μM) to NT-647-labeled clusterin (25 nM) was evaluated in fluid phase by microscale themophoresis. Thermophoresis was recorded at 50% LED power and 80% MST power for 30 s in a Monolith NT.115 instrument. The relative fluorescence in the thermophoresis phase of the experiment was plotted against the concentration of Lpd.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: SDS Page, Silver Staining, Marker, Incubation, Negative Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Labeling, Fluorescence, Concentration Assay

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A , Localization of the region within vitronectin that mediates binding to Lpd. Vitronectin uses one contact region i.e. aa 354–363 to contact Lpd. Vitronectin 80–396 (Vn 80-396 ) and seven deletion mutants were expressed in HEK cells and purified. The numbers refer to amino acids residues that are included in each construct ( left panel ). Black indicates the heparin binding regions of Vitronectin ( left panel ). Binding of serum purified full-length vitronectin and vitronectin deletion mutants (5 μg/ml) to immobilized Lpd was assayed by ELISA ( right panel ). Bound vitronectin was detected with polyclonal vitronectin antiserum followed by HRP-conjugated anti-rabbit. B , Heparin inhibits binding of vitronectin to Lpd and the effect was dose-dependent. The effect of heparin (0.01–5 mg/ml) on vitronectin binding to immobilized Lpd was assayed. Bound vitronectin was detected with polyclonal vitronectin antiserum and HRP-conjugated anti-rabbit pAb. C , Schematic picture of full length Lpd and its fragments. D , The vitronectin-binding regions are located within two separate binding domains of Lpd. Equimolar amounts (13.3 nM) of full length Lpd and Lpd deletion mutants were immobilized onto microtiter plates, vitronectin was added and bound vitronectin was quantified. E , The clusterin-binding regions are located within two separate binding domains of Lpd. Equimolar amounts (13.3 nM) of full length Lpd and Lpd deletion mutants were immobilized onto microtiter plates, clusterin was added and bound clusterin was quantified. The mean values of three independent experiments and SD are presented. Statistical significance of differences was estimated using Student’s t test. *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: Binding Assay, Purification, Construct, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: A, Effect of increasing clusterin levels in the presence of a constant concentration of vitronectin. Binding of clusterin (used at the indicated concentrations) and vitronectin (5 μg/ml) to immobilized Lpd was analysed by ELISA. Bound clusterin was detected with anti-clusterin mAb (■) and bound vitronectin was detected with anti-vitronectin mAb (◇). B, In a reverse setting, the clusterin concentration was kept constant (2.5 μg/ml) and binding of vitronectin (used at the indicated concentrations) was evaluated. The mean values of three independent experiments and SD are presented.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

doi: 10.1371/journal.pone.0137630

Figure Lengend Snippet: Both vitronectin ( A ) and clusterin ( B ) when bound to immobilized Lpd inhibits C5b-9 deposition. A , Vitronectin (10–50 μg/ml) or Factor H (10–50 μg/ml) was bound to immobilized Lpd and after extensive washing C5b-6 and C7 were added. After 10 min incubation C8 and C9 were added and C5b-9 deposition was detected with mouse anti-C5b-9 mAb and HRP-conjugated anti-mouse pAb. B , Clusterin (2.5–20 μg/ml) or Factor H (2.5–20 μg/ml) was bound to immobilized Lpd and after extensive washing C5b-6 and C7 were added. After 10 min incubation C8 and C9 were added and C5b-9 deposition was detected with mouse anti-C5b-9 mAb and HRP-conjugated anti-mouse pAb. The mean values of three independent experiments and SD are presented. Statistical significance of differences was estimated using Student’s t test. ***, p ≤ 0.001.

Article Snippet: Polyclonal rabbit anti-human vitronectin and polyclonal goat anti-Factor H were obtained from Complement Technology (Tyler, TE) and polyclonal goat anti-plasminogen from Acris Antibodies GmbH (Herford, Germany).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Repeating Structures of the Major Staphylococcal Autolysin Are Essential for the Interaction with Human Thrombospondin 1 and Vitronectin *

doi: 10.1074/jbc.M113.521229

Figure Lengend Snippet: Human TSP-1 binds preferentially to repeats R1ab-R2ab as shown by surface plasmon resonance studies. A, human TSP-1 (0.1 μg) was immobilized on 96-well plates (MaxiSorp) and incubated with various molecular ratios of AtlE R1ab-R2ab or AtlE R1ab. The binding of repeats was detected using a polyclonal anti-AtlE-R1ab-R2ab IgG followed by incubation with a peroxidase-coupled secondary antibody. Results are expressed as means ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001; ns, not significant. B, surface plasmon resonance sensorgrams of heterologously expressed AtlE R1ab-R2ab show a dose-dependent binding to immobilized hTSP-1. A CM5 biosensor was coated with hTSP-1 (∼4000 response units), and the heterologously expressed repeats R1ab-R2ab of AtlE were used as analytes. The values of the control flow cells were subtracted from each sensorgram. C, surface plasmon resonance sensorgrams of heterologously expressed AtlE R1ab-2ab show a dose-dependent binding to immobilized human vitronectin. Vn was immobilized on the CM5 biosensor (∼2500 response units), and the heterologously expressed repeats R1ab-R2ab of AtlE were used as analytes. The values of the control flow cells were subtracted from each sensorgram. D, low binding activity of heterologously expressed AtlE repeat R1ab (25 μg/ml) to immobilized hTSP-1 as analyzed by an SPR study. Shown is an SPR sensorgram of a manual run.

Article Snippet: Contaminations with fibronectin or vitronectin were excluded by immunoblot analysis of purified hTSP-1with primary antibodies against vitronectin (rabbit anti-human vitronectin, CompTech) and fibronectin (rabbit anti-human fibronectin, Dako) and a secondary goat anti-rabbit IgG coupled to alkaline phosphatase (1:7500, Promega).

Techniques: SPR Assay, Incubation, Binding Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Repeating Structures of the Major Staphylococcal Autolysin Are Essential for the Interaction with Human Thrombospondin 1 and Vitronectin *

doi: 10.1074/jbc.M113.521229

Figure Lengend Snippet: Human TSP-1 and vitronectin bind dose-dependently to the R1ab-R2ab repeats of Atl and compete for binding. A, binding of hTSP-1 to immobilized Atl repeats R1ab-R2ab. The heterologously expressed repeats R1ab-R2ab (0.5 μg) were immobilized on microtiter plates (MaxiSorp) and incubated with increasing amounts of hTSP-1. B, binding of human vitronectin to immobilized Atl repeats R1ab-R2ab. The heterologously expressed repeats R1ab-R2ab (0.5 μg) were immobilized on microtiter plates (MaxiSorp) and incubated with increasing amounts of Vn. C and D, human TSP-1 competes with human vitronectin for binding to the immobilized Atl repeats R1ab-R2ab. The heterologously expressed repeats R1ab-R2ab (0.5 μg) were immobilized on microtiter plates (MaxiSorp) and incubated with hTSP-1 (1000 ng/well) in the presence of increasing molecular ratios of Vn (C) or with Vn (125 ng/well) in the presence of increasing molecular ratios of hTSP-1 (D). Bound hTSP was detected using a polyclonal mouse anti-hTSP-1 IgG antibody followed by incubation with a peroxidase-coupled secondary anti-mouse antibody, and bound Vn was detected using a polyclonal rabbit anti-Vn IgG followed by incubation with a peroxidase-coupled secondary anti-rabbit antibody. Results are expressed as means ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; ns, not significant.

Article Snippet: Contaminations with fibronectin or vitronectin were excluded by immunoblot analysis of purified hTSP-1with primary antibodies against vitronectin (rabbit anti-human vitronectin, CompTech) and fibronectin (rabbit anti-human fibronectin, Dako) and a secondary goat anti-rabbit IgG coupled to alkaline phosphatase (1:7500, Promega).

Techniques: Binding Assay, Incubation