Journal: The Journal of Biological Chemistry

Article Title: Thromboxane Receptor Signaling Is Required for Fibronectin-induced Matrix Metalloproteinase 9 Production by Human and Murine Macrophages and Is Attenuated by the Arhgef1 Molecule *

doi: 10.1074/jbc.M111.282772

Figure Lengend Snippet: Human alveolar macrophages are induced to express MMP9 when cultured on fibronectin. A, induction of MMP9 expression by human macrophages cultured on plastic (open bars) or fibronectin (closed bars) as measured by qPCR at the indicated cell concentrations. MMP9 expression on fibronectin is shown as fold overexpression of cells cultured on plastic at each respective concentration from the same individual. The results are compiled from cells obtained from four individuals. The data represent the means ± S.E. B, representative gelatin zymogram of conditioned media from human macrophages cultured on either plastic (ctl) or FN at the indicated cell concentrations. C, quantitation of MMP9 activity in conditioned media from cells cultured on FN or plastic (ctl) at 0.125 × 106 cells/ml. The results are compiled from cells obtained from six individuals. The data represent the means ± S.E. *, p < 0.05 Student's two-tailed t test compared with cells cultured on plastic.

Article Snippet: Tissue-culture 96-well plates (Costar high binding catalogue number 3590) were coated with 10 μg/ml of murine fibronectin (Molecular Innovations, Nori, MN; catalogue number MFBN) or human fibronectin (Molecular Innovations; catalogue number HFBN) in Dulbecco's PBS without Ca 2+ or Mg 2+ (Mediatech, Inc., Manassas, VA) for 2 h at room temperature.

Techniques: Cell Culture, Expressing, Over Expression, Concentration Assay, Quantitation Assay, Activity Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Thromboxane Receptor Signaling Is Required for Fibronectin-induced Matrix Metalloproteinase 9 Production by Human and Murine Macrophages and Is Attenuated by the Arhgef1 Molecule *

doi: 10.1074/jbc.M111.282772

Figure Lengend Snippet: Human alveolar macrophages produce TXB2 when cultured on fibronectin and thromboxane receptor signaling is required for MMP9 production. A, TXB2 production measured in conditioned media from human macrophages cultured on fibronectin (closed bars) or plastic (open bars) at the indicated cellular concentrations. B, TXB2 measured in conditioned media from macrophages cultured at 0.125 × 106 cells/ml on either plastic (ctl) or FN from four individuals. The data represent the means ± S.E. C, gelatin zymogram of conditioned media from cells cultured as indicated and in the presence of increasing doses of the thromboxane receptor antagonist PTA2 (at 1.25, 6.25, and 25 μm, respectively). Below the zymogram is quantitation of MMP9 activity by densitometric analysis.

Article Snippet: Tissue-culture 96-well plates (Costar high binding catalogue number 3590) were coated with 10 μg/ml of murine fibronectin (Molecular Innovations, Nori, MN; catalogue number MFBN) or human fibronectin (Molecular Innovations; catalogue number HFBN) in Dulbecco's PBS without Ca 2+ or Mg 2+ (Mediatech, Inc., Manassas, VA) for 2 h at room temperature.

Techniques: Cell Culture, Quantitation Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Thromboxane Receptor Signaling Is Required for Fibronectin-induced Matrix Metalloproteinase 9 Production by Human and Murine Macrophages and Is Attenuated by the Arhgef1 Molecule *

doi: 10.1074/jbc.M111.282772

Figure Lengend Snippet: Human peripheral blood monocytes cultured on fibronectin are induced to express MMP9 and that is dependent on thromboxane receptor signaling. A, MMP9 induction was measured by qPCR at the indicated cell concentrations. MMP9 expression on fibronectin (closed bars) is represented as fold overexpression of cells cultured on plastic at each respective concentration from the same individual. The results are compiled from cells obtained from two individuals. The data represent the means ± S.E. B, representative zymogram of conditioned media from monocytes cultured on either plastic (ctl) or FN at the indicated cellular concentrations. C, MMP9 production as measured by ELISA in conditioned media from monocytes cultured on plastic (ctl) or FN at 0.25 × 106 cells/ml. The results are compiled from cells obtained from eight individuals. The data represent the means ± S.E. D, TXB2 production in conditioned media from monocytes cultured on either plastic or FN at the indicated cellular concentrations. The results are compiled from cells obtained from three individuals. The data represent the means ± S.E. E, TXB2 in conditioned media from monocytes cultured on either plastic (ctl) or FN at 0.25 × 106 cells/ml. The results are compiled from cells obtained from 10 individuals. The data represent the means ± S.E. F, MMP9 levels as measured by ELISA in conditioned media from monocytes cultured as indicated with increasing concentrations of the thromboxane receptor antagonist L-655,240 (1.75, 7, 28, and 112 μm). G, MMP9 as measured by ELISA in conditioned media from monocytes cultured as indicated with the thromboxane receptor antagonist PTA2 (7.5 μm). The results are compiled from cells obtained from six individuals. *, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on plastic. #, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on fibronectin.

Article Snippet: Tissue-culture 96-well plates (Costar high binding catalogue number 3590) were coated with 10 μg/ml of murine fibronectin (Molecular Innovations, Nori, MN; catalogue number MFBN) or human fibronectin (Molecular Innovations; catalogue number HFBN) in Dulbecco's PBS without Ca 2+ or Mg 2+ (Mediatech, Inc., Manassas, VA) for 2 h at room temperature.

Techniques: Cell Culture, Expressing, Over Expression, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Thromboxane Receptor Signaling Is Required for Fibronectin-induced Matrix Metalloproteinase 9 Production by Human and Murine Macrophages and Is Attenuated by the Arhgef1 Molecule *

doi: 10.1074/jbc.M111.282772

Figure Lengend Snippet: Fibronectin induces macrophages to produce MMP9, PGE2, and TXB2. A, Mmp9 expression was measured by qPCR in peritoneal macrophages cultured on FN (10 μg/ml) or plastic (ctl) plated at the indicated cellular concentrations and cultured for 48 h. Mmp9 expression is shown on a log scale as fold induction over expression of wild type cells cultured on plastic (ctl) at each respective concentration. The number of experiments at each concentration (0.03, 0.13, 0.5, and 2.0 × 106 cells/ml) for wild type cells on plastic (open bars) are represented by n = 3, 5, 10, and 3, respectively; wild type cells on fibronectin (gray bars) are represented by n = 3, 12, 12, and 3; Arhgef1−/− cells on plastic (hatched bars) are represented by n = 3, 6, 11, and 3; Arhgef1−/− cells on fibronectin (black bars) are represented by n = 3, 12, 12, and 3. The data represent the means ± S.E. B, representative zymogram of conditioned media from peritoneal macrophages cultured on either plastic (ctl) or FN at the indicated cellular concentrations from wild type (+/+) and Arhgef1−/− (−/−) samples. Molecular weight standards and respective enzymatic activities of MMP9 and MMP2 are shown. C, quantitation of MMP9 activity as determined by densitometric analyses of zymograms. MMP9 activity is shown in arbitrary units and represents n = 7 for wild type cells on plastic (open bars) and on fibronectin (gray bars) at all cellular concentrations. For Arhgef1−/− samples, n = 6 for cells on plastic (hatched bars) and on fibronectin (black bars) at all cellular concentrations. The data represent the means ± S.E. D, PGE2 was measured by ELISA in conditioned media from macrophages cultured for 48 h on either plastic or fibronectin. For wild type (open bars), n = 4, and for Arhgef1−/− (black bars), n = 6. The data represent the means ± S.E. The dotted line indicates the limit of detection. E, TXB2 was measured by ELISA in conditioned media from macrophages cultured for 48 h on either plastic or fibronectin. For wild type (open bars), n = 4, and for Arhgef1−/− (black bars), n = 6. The data represent the means ± S.E. The dotted line indicates the limit of detection. *, p < 0.05 Student's two-tailed t test comparing MMP9 expression/activity on fibronectin to respective cells on plastic. #, p < 0.05 Student's two-tailed t test comparing wild type to Arhgef1−/− cells under identical conditions.

Article Snippet: Tissue-culture 96-well plates (Costar high binding catalogue number 3590) were coated with 10 μg/ml of murine fibronectin (Molecular Innovations, Nori, MN; catalogue number MFBN) or human fibronectin (Molecular Innovations; catalogue number HFBN) in Dulbecco's PBS without Ca 2+ or Mg 2+ (Mediatech, Inc., Manassas, VA) for 2 h at room temperature.

Techniques: Expressing, Cell Culture, Over Expression, Concentration Assay, Molecular Weight, Quantitation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Thromboxane Receptor Signaling Is Required for Fibronectin-induced Matrix Metalloproteinase 9 Production by Human and Murine Macrophages and Is Attenuated by the Arhgef1 Molecule *

doi: 10.1074/jbc.M111.282772

Figure Lengend Snippet: Cyclooxygenase activity but not EP4 receptor signaling is required for MMP9 production by murine macrophages cultured on fibronectin. A, representative MMP9 gelatin zymograms of conditioned media from peritoneal macrophages cultured on either plastic (ctl) or FN and treated with increasing concentrations of aspirin (1.25, 5.0, and 20 mm, respectively). Wild type (+/+) and Arhgef1−/− (−/−) samples from separate zymograms are shown. Molecular weight standards and respective enzymatic activities of MMP9 and MMP2 are shown. B, quantitation of MMP9 activity as determined by densitometric analysis of zymograms in A. MMP9 activity is shown in arbitrary units and represents n = 6 for both wild type (open bars) and Arhgef1−/− (black bars) samples. The results are compiled from two independent experiments. The data represent the means ± S.E. *, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on plastic. #, p < 0.05 Student's two-tailed t test comparing conditioned media from wild type cells to Arhgef1−/− cells cultured under identical conditions. $, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on fibronectin. C, quantitation of MMP9 activity in conditioned media from macrophages cultured on fibronectin in the presence of the EP4 antagonist L161,982 (10 μm). MMP9 activity was normalized to fibronectin response for each genotype. The results are compiled from two independent experiments with wild type (open bars, n = 5) and Arhgef1−/− (black bars, n = 5) samples. The data represent the means ± S.E. *, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on plastic. #, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on fibronectin.

Article Snippet: Tissue-culture 96-well plates (Costar high binding catalogue number 3590) were coated with 10 μg/ml of murine fibronectin (Molecular Innovations, Nori, MN; catalogue number MFBN) or human fibronectin (Molecular Innovations; catalogue number HFBN) in Dulbecco's PBS without Ca 2+ or Mg 2+ (Mediatech, Inc., Manassas, VA) for 2 h at room temperature.

Techniques: Activity Assay, Cell Culture, Molecular Weight, Quantitation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Thromboxane Receptor Signaling Is Required for Fibronectin-induced Matrix Metalloproteinase 9 Production by Human and Murine Macrophages and Is Attenuated by the Arhgef1 Molecule *

doi: 10.1074/jbc.M111.282772

Figure Lengend Snippet: Thromboxane receptor signaling is necessary for fibronectin-induced MMP9 production by macrophages and is attenuated by Arhgef1. A, representative gelatin zymograms of conditioned media from macrophages cultured on plastic (ctl) or FN and treated with 1.56 or 6.25 μm of the thromboxane receptor antagonist PTA2. Wild type (+/+) and Arhgef1−/− (−/−) samples from separate zymograms are shown. B, quantitation of MMP9 activity as determined by densitometric analysis of zymograms in A. The results are representative of two independent experiments. C, representative zymograms from conditioned media from macrophages cultured on plastic or fibronectin and treated with 28 μm of the thromboxane receptor antagonist L-655,240. D, quantitation of MMP9 activity as determined by densitometric analysis of zymograms in C. The results are compiled from two independent experiments with n = 4 for both wild type (open bars) and Arhgef1−/− (black bars) samples. The data represent the means ± S.E. *, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on plastic. #, p < 0.05 Student's two-tailed t test comparing conditioned media from wild type cells to Arhgef1−/− cells cultured under identical conditions. $, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on fibronectin. E, MMP2 activity was quantitated in conditioned media as previously described for MMP9. The results are compiled from four independent experiments with n = 10 for wild type (open bars) and Arhgef1−/− (black bars) samples cultured on either plastic or fibronectin. For cells treated with 20 mm aspirin, n = 6 for wild type (open bars) and Arhgef1−/− (black bars) samples. For cells treated with 28 μm L-655,240, n = 4 for wild type (open bars) and Arhgef1−/− (black bars) samples. The data represent the means ± S.E. F, MMP9 activity was quantitated as previously described and normalized to a percentage of fibronectin response for each genotype. The cells were either untreated or treated with 0.5% DMSO or 28 μm L-655,240. The results are compiled from two independent experiments with n = 3 for all conditions and genotypes. The data represent the means ± S.E. *, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on fibronectin. G, relative Mmp9 expression was measured by qPCR in macrophages cultured on fibronectin in the presence of the thromboxane receptor agonist (U-46619, 10 nm), S1P (40 nm), or LPA (10 μm). Mmp9 expression is displayed as a percentage of fibronectin response for each genotype. Wild type (open bars, n = 12, 6, 3, and 3 for fibronectin, +U-46619, +S1P, and +LPA, respectively) and Arhgef1−/− (black bars, n = 14, 6, 3, and 2, respectively) from at least two independent experiments. The data represent the means ± S.E. *, p < 0.05 Student's two-tailed t test compared with conditioned media from cells cultured on fibronectin. #, p < 0.05 Student's two-tailed t test comparing conditioned media from wild type cells to Arhgef1−/− cells cultured under identical conditions.

Article Snippet: Tissue-culture 96-well plates (Costar high binding catalogue number 3590) were coated with 10 μg/ml of murine fibronectin (Molecular Innovations, Nori, MN; catalogue number MFBN) or human fibronectin (Molecular Innovations; catalogue number HFBN) in Dulbecco's PBS without Ca 2+ or Mg 2+ (Mediatech, Inc., Manassas, VA) for 2 h at room temperature.

Techniques: Cell Culture, Quantitation Assay, Activity Assay, Two Tailed Test, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Thromboxane Receptor Signaling Is Required for Fibronectin-induced Matrix Metalloproteinase 9 Production by Human and Murine Macrophages and Is Attenuated by the Arhgef1 Molecule *

doi: 10.1074/jbc.M111.282772

Figure Lengend Snippet: ARHGEF1 expression negatively correlates with MMP9 production by monocytes cultured on fibronectin. Relative ARHGEF1 expression as measured by RT-PCR is expressed on the x axis. ARHGEF1 expression was normalized to GAPDH expression and displayed relative to the lowest expressing individual. MMP9 production was measured in conditioned media by ELISA and is expressed on the y axis. Shown are the results from monocytes obtained from nine individuals cultured under identical conditions where each point represents the values obtained from a separate individual. A Pearson product moment correlation analysis was performed, and a correlation coefficient of −0.737 was obtained with a p = 0.0234 between ARHGEF1 expression and MMP9 production. The dotted lines denote bivariate normal ellipse for 95% of the values. The solid line represents the linear fit.

Article Snippet: Tissue-culture 96-well plates (Costar high binding catalogue number 3590) were coated with 10 μg/ml of murine fibronectin (Molecular Innovations, Nori, MN; catalogue number MFBN) or human fibronectin (Molecular Innovations; catalogue number HFBN) in Dulbecco's PBS without Ca 2+ or Mg 2+ (Mediatech, Inc., Manassas, VA) for 2 h at room temperature.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity

doi: 10.1074/jbc.m115.691097

Figure Lengend Snippet: FIGURE 5. Comparative degradation rates and fragmentation patterns of human fibronectin in the presence of KLK2e (lanes 2–6) glyco-KLK2 (lanes 7–11) as determined by SDS-PAGE. Aliquots were taken at represen- tative intervals and analyzed by SDS-PAGE followed by Coomassie Blue stain- ing. Fibronectin alone is stable at 37 °C for at least 24 h. Overall, the break- downbyglyco-KLK2appearsmoreefficientandresultsinsomeuniquebands after 24-h incubation, as indicated by arrows.

Article Snippet: For fibronectin degradation, 50 g of purified human serum fibronectin (Innovative Research, Novi, MI) were incubated with 0.5 g of KLK2 from E. coli or LEXSY cells for different periods of time (0, 1, 2, 4, and 24 h) at 37 °C in 50 mM Tris-HCl, pH 8.0, containing 0.1% Nonidet P-40, 150 mM NaCl.

Techniques: SDS Page, Staining, Incubation

Journal: Cell

Article Title: Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading

doi: 10.1016/j.cell.2019.04.037

Figure Lengend Snippet: Key Resources Table

Article Snippet: Human Plasma Fibronectin Purified Protein , Merck (Sigma Aldrich) , Cat# FC010.

Techniques: Purification, Transduction, Recombinant, Avidin-Biotin Assay, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Mutagenesis, Derivative Assay, Construct, shRNA, Software

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: The effect of PIP knockdown on ERK-Akt and integrin-β1signaling . (A) Western blot analysis to measure the levels of phosphorylated (Ph)-ERK, total (T)-ERK, ph-Akt, and T-Akt following PIP-knockdown with siRNA duplex1 (PIP-D1) and duplex2 (PIP-D2) in MDA-MB-453 cell line. Fold changes of Phospho/Total ratios (Ph/T-RR) were assessed relative to non-targeting siRNA control (CTL). (B) Western blot analysis to measure the level of ph-CREB1, T-CREB1, and ILK1 following PIP-knockdown as described in (A). Ph-ATF1 is the phosphorylated form of CREB-related protein that is known to be detected by this antibody. (C) Integrin-β1 immunoprecipitation (IP). IP assays were carried out with Integrin-β1 following PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Non-targeting siRNA was used as control (CTL). Western blot analysis was carried out on IP samples to measure the integrin-β1 binding to ILK1 and ErbB2. Immunoblotting with integrin-β1 antibody was used as a loading control. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown were measured relative to that of control-siRNA. (D) Integrin-β1 immunoprecipitation following PIP-knockdown and the addition of fibronectin fragments (Fn-fs). PIP-knockdown with PIP-D1 was carried out as described in (C). Twenty-four hours after PIP-knockdown, cells were treated with α-chymotryptic fibronectin fragment 120K at 100 µg/ml concentration. Control cells were treated with vehicle only. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown + Fn-fs were measured relative to the control. (E) The effect of fibronectin fragments on cell invasion following PIP-knockdown. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. Treatment with fibronectin fragments was carried out as described in (D). Error Bars: ± 2SEM. Δ; is the difference between CTL and PIP-D1+Fn-fs groups. ERK, extracellular signal-regulated kinase; ILK1, integrin-linked kinase 1; RR, relative risk; SEM, standard error of the mean.

Article Snippet: Treatment with Purified Human Fibronectin (α-Chymotryptic Fragment 120K, Merck Millipore) at 100 μg/ml concentration was carried out 24 hours after PIP-knockdown.

Techniques: Knockdown, Western Blot, Control, Immunoprecipitation, Binding Assay, Concentration Assay, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Schematic diagram of the PIP signaling pathway in ER-negative breast cancer . Red arrow denotes stimulatory effect. ER, estrogen receptor; Fn, fibronectin; Fn-f, Fibronectin fragment; ITG-β1, integrin-β1.

Article Snippet: Treatment with Purified Human Fibronectin (α-Chymotryptic Fragment 120K, Merck Millipore) at 100 μg/ml concentration was carried out 24 hours after PIP-knockdown.

Techniques:

Journal:

Article Title: Isolation of Enterococcus faecalis Clinical Isolates That Efficiently Adhere to Human Bladder Carcinoma T24 Cells and Inhibition of Adhesion by Fibronectin and Trypsin Treatment

doi:

Figure Lengend Snippet: Adherence of fibronectin-treated (A) and untreated (B) E. faecalis strains to T24 cells. The efficiently adhesive strains AS11 and AS12 were treated with fibronectin for 1 h at 37°C and then examined for the adherence to T24 cells.

Article Snippet: Fibronectin (purified by affinity chromatography of human plasma on gelatin-Sepharose columns) and fibrinogen (purified from human plasma) were purchased from Koken (Tokyo, Japan).

Techniques:

Journal:

Article Title: Isolation of Enterococcus faecalis Clinical Isolates That Efficiently Adhere to Human Bladder Carcinoma T24 Cells and Inhibition of Adhesion by Fibronectin and Trypsin Treatment

doi:

Figure Lengend Snippet: Adherence of untreated strains AS11 (A) and AS12 (B) and of fibronectin-treated strains AS11 (C) and AS12 (D) to T24 cells.

Article Snippet: Fibronectin (purified by affinity chromatography of human plasma on gelatin-Sepharose columns) and fibrinogen (purified from human plasma) were purchased from Koken (Tokyo, Japan).

Techniques:

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Plasma and recombinant VWF bind to fibronectin. Human fibronectin was captured on a plate and used to bind plasma VWF (healthy controls or subjects with type 3 VWD) or recombinant VWF. Decreased binding was noted for VWF constructs unable to form multimeric structures (2773R and 87S) as well as constructs with VWF A1 domain variants (1392A, 1395A, 1399H). Results are graphed as a ratio of VWF bound to fibrinogen over VWF antigen to account for minor differences in total protein. Error bars show 1 standard deviation. Results are average of ≥3 experiments. * signifies P <.05 and ** signifies P <.02 (plasma samples compared against each other, recombinant samples compared to WT recombinant VWF). VWD, von Willebrand disease; VWF, von Willebrand factor; VWF:Ag, von Willebrand factor antigen; WT, wild‐type

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Clinical Proteomics, Recombinant, Binding Assay, Construct, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Fibronectin preferentially interacts with higher‐molecular‐weight VWF multimers. Binding of VWF to human fibronectin is shown using purified preparations of recombinant ultra‐high‐molecular‐weight VWF multimers (ultra), high‐molecular‐weight VWF multimers (high), medium‐molecular‐weight VWF multimers (medium), and low‐molecular‐weight VWF multimers (low). Results are graphed as a fraction of WT recombinant VWF binding with all multimers present to normalize for the total amount of VWF present in each preparation. Error bars show 1 standard deviation. Results are average of ≥3 experiments. VWF, von Willebrand factor; VWF:Ag, von Willebrand factor antigen; WT, wild‐type

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Molecular Weight, Binding Assay, Purification, Recombinant, High Molecular Weight, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Inhibition of VWF‐fibronectin interactions. VWF binding to human fibronectin was measured alone (WT), and in the presence of anti‐VWF antibody AVW‐3, which blocks the A1 domain (anti‐VWF A1), a polyclonal VWF antibody (polyclonal anti‐VWF), collagen type III and IV, and extracellular matrix proteins thrombospondin, vitronectin, and laminin. Results are graphed as a percent of WT VWF bound to fibronectin in the absence of antibody. Error bars show 1 standard deviation. Results are average of three or more experiments. ** signifies P < .01. ECM, extracellular matrix; VWF, von Willebrand factor; VWF:Ag, von Willebrand factor antigen; WT, wild‐type

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Inhibition, Binding Assay, Standard Deviation

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: Binding affinities of VWF for collagen IV and fibronectin

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Binding Assay, Concentration Assay

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Fibronectin binding to von Willebrand factor occurs via the A1 domain

doi: 10.1002/rth2.12534

Figure Lengend Snippet: VWF binding to fibronectin and collagen IV. VWF binding to human fibronectin (black) and human collagen IV (gray) was measured using the Octet Biosensor to determine binding affinities. The x axis shows varying VWF concentrations from 0 to 3 μg/mL, and the y axis shows the K D in nM. K D , dissociation constant; VWF, von Willebrand factor

Article Snippet: Amine binding maleic anhydride plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg/mL with human purified plasma‐derived fibronectin (Haematologic Technologies, Inc., Essex Junction, VT, USA) diluted in phosphate‐buffered saline (PBS; pH 7.4) and incubated at 2 to 4°C overnight.

Techniques: Binding Assay