heparinase iii  (R&D Systems)

 
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    Name:
    Recombinant P heparinus Heparinase III Protein CF
    Description:
    The Recombinant P heparinus Heparinase III Protein from R D Systems is derived from E coli The Recombinant P heparinus Heparinase III Protein has been validated for the following applications Enzyme Activity
    Catalog Number:
    6145-gh-010
    Price:
    269
    Applications:
    Enzyme Activity
    Purity:
    >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie« Blue stain at 5 ╡g per lane.
    Conjugate:
    Unconjugated
    Size:
    10 ug
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant P. heparinus Heparinase III Protein
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    Structured Review

    R&D Systems heparinase iii
    Recombinant P heparinus Heparinase III Protein CF
    The Recombinant P heparinus Heparinase III Protein from R D Systems is derived from E coli The Recombinant P heparinus Heparinase III Protein has been validated for the following applications Enzyme Activity
    https://www.bioz.com/result/heparinase iii/product/R&D Systems
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    heparinase iii - by Bioz Stars, 2020-11
    94/100 stars

    Images

    1) Product Images from "Biomimetic post-capillary venule expansions for leukocyte adhesion studies"

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27566-z

    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Figure Legend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Techniques Used:

    2) Product Images from "Biomimetic post-capillary venule expansions for leukocyte adhesion studies"

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27566-z

    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Figure Legend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Techniques Used:

    3) Product Images from "Biomimetic post-capillary venule expansions for leukocyte adhesion studies"

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27566-z

    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Figure Legend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Techniques Used:

    4) Product Images from "Calcitriol Imparts Neuroprotection In Vitro to Midbrain Dopaminergic Neurons by Upregulating GDNF Expression"

    Article Title: Calcitriol Imparts Neuroprotection In Vitro to Midbrain Dopaminergic Neurons by Upregulating GDNF Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062040

    Calcitriol is neuroprotective and does not increase differentiation. The increase in dopamine neurons obtained from cultures was shown to be a result of increased neuroprotection afforded through calcitriol addition, rather than changes in differentiation. (A–D) Immunolabeling with a pan-caspase marker (red) and antibodies to tyrosine hydroxylase (green) counterstained with DAPI (blue), for: A) control media, B) 10 nM calcitriol, C) 0.3 U/ml Heparinase III and D) 10 nM calcitriol with 0.3 U/ml heparinase III. Scale bars: 20 µM. E) The number of apoptotic dopamine neurons was significantly increased when heparinase III was added to media. Bars represent the ratio of caspase+ dopamine neurons to caspase+ DAPI+ cells. Error bars represent SEM, *** p
    Figure Legend Snippet: Calcitriol is neuroprotective and does not increase differentiation. The increase in dopamine neurons obtained from cultures was shown to be a result of increased neuroprotection afforded through calcitriol addition, rather than changes in differentiation. (A–D) Immunolabeling with a pan-caspase marker (red) and antibodies to tyrosine hydroxylase (green) counterstained with DAPI (blue), for: A) control media, B) 10 nM calcitriol, C) 0.3 U/ml Heparinase III and D) 10 nM calcitriol with 0.3 U/ml heparinase III. Scale bars: 20 µM. E) The number of apoptotic dopamine neurons was significantly increased when heparinase III was added to media. Bars represent the ratio of caspase+ dopamine neurons to caspase+ DAPI+ cells. Error bars represent SEM, *** p

    Techniques Used: Immunolabeling, Marker

    5) Product Images from "Biomimetic post-capillary venule expansions for leukocyte adhesion studies"

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27566-z

    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Figure Legend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Techniques Used:

    6) Product Images from "Syndecan-4 mediates macrophage uptake of group V secretory phospholipase A2-modified LDL"

    Article Title: Syndecan-4 mediates macrophage uptake of group V secretory phospholipase A2-modified LDL

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M800450-JLR200

    Macrophage uptake of group V sPLA2-modified LDL (GV-LDL) depends on cellular proteoglycans. J-774 cells were preincubated for 4 h in the presence or absence of 1 U/ml heparinase III + 0.15 U/ml chondroitinase ABC in PBS containing 0.1 M sodium acetate
    Figure Legend Snippet: Macrophage uptake of group V sPLA2-modified LDL (GV-LDL) depends on cellular proteoglycans. J-774 cells were preincubated for 4 h in the presence or absence of 1 U/ml heparinase III + 0.15 U/ml chondroitinase ABC in PBS containing 0.1 M sodium acetate

    Techniques Used: Modification

    Related Articles

    other:

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies
    Article Snippet: All inhibitors were added just before the experiment start, except for Heparinase III which was added to the initial coating solution.

    Immunofluorescence:

    Article Title: Syndecan-4 mediates macrophage uptake of group V secretory phospholipase A2-modified LDL
    Article Snippet: .. J-774 cells were treated for 4 h with 1 U/ml heparinase III and 0.15 U/ml chondroitinase ABC and subjected to indirect immunofluorescence using goat anti-mouse syndecan-3 IgG (R and D Systems, Minneapolis, MN) or rat anti-mouse syndecan-4 IgG (BD Biosciences, Franklin Lakes, NJ). .. Alexa-Fluor 488-labeled rabbit anti-goat and goat anti-rat IgGs (Molecular Probes) were used as secondary antibodies at a dilution of 1:200.

    Blocking Assay:

    Article Title: Calcitriol Imparts Neuroprotection In Vitro to Midbrain Dopaminergic Neurons by Upregulating GDNF Expression
    Article Snippet: .. Heparinase III (R & D Systems) was used to block GDNF signaling , , by adding 0.3 U/ml to culture media. ..

    SDS Page:

    Article Title: Prevention of TGFβ induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr−/− mice 1 mice 1 [S]
    Article Snippet: .. Equal amounts of protein were treated with chondroitinase or heparinase III to remove GAG, then separated by SDS-PAGE using 8% gels (biglycan molecular mass 42 kDa; perlecan molecular mass 400 kDa) followed by immunoprobing with an anti-biglycan antibody (R and D Systems) or anti-perlecan antibody (Accurate Chemical, Westbury, NY). .. Blotting for β-actin (Sigma, St. Louis, MO; AF2066) served as a loading control.

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    R&D Systems heparinase iii
    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and <t>Heparinase</t> <t>III.</t> Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Heparinase Iii, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparinase iii/product/R&D Systems
    Average 94 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    heparinase iii - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

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    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Journal: Scientific Reports

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    doi: 10.1038/s41598-018-27566-z

    Figure Lengend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Article Snippet: All inhibitors were added just before the experiment start, except for Heparinase III which was added to the initial coating solution.

    Techniques:

    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Journal: Scientific Reports

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    doi: 10.1038/s41598-018-27566-z

    Figure Lengend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Article Snippet: Heparinase III (R & D Systems) was used at 40 micrograms per milliliter.

    Techniques:

    Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Article Snippet: In some experiments, the exosomes were treated with bacterial heparitinase and probed for syndecan-1 (R & D Systems).

    Techniques: Isolation, Purification, Enzyme-linked Immunosorbent Assay, Derivative Assay, Flow Cytometry, Cytometry, Affinity Purification, Western Blot

    Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Article Snippet: In some experiments, the exosomes were treated with bacterial heparitinase and probed for syndecan-1 (R & D Systems).

    Techniques: Purification, Magnetic Beads, Flow Cytometry, Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

    Model of exosome-target cell interaction mediated by fibronectin. Step 1 , fibronectin ( FN ) is captured on the surface of exosomes by heparan sulfate proteoglycans ( HSPG ). Step 2 , fibronectin, on exosomes binds to heparan sulfate ( HS ) on the surface of the target cell. Step 3 , removal of exosome heparan sulfate or cell surface heparan sulfate with heparitinase, addition of exogenous heparin or heparin mimetics, addition of Hep-II domain-containing fragment of fibronectin, or exposure to antibody against the Hep-II domain of fibronectin dramatically diminishes exosome interaction with target cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Model of exosome-target cell interaction mediated by fibronectin. Step 1 , fibronectin ( FN ) is captured on the surface of exosomes by heparan sulfate proteoglycans ( HSPG ). Step 2 , fibronectin, on exosomes binds to heparan sulfate ( HS ) on the surface of the target cell. Step 3 , removal of exosome heparan sulfate or cell surface heparan sulfate with heparitinase, addition of exogenous heparin or heparin mimetics, addition of Hep-II domain-containing fragment of fibronectin, or exposure to antibody against the Hep-II domain of fibronectin dramatically diminishes exosome interaction with target cells.

    Article Snippet: In some experiments, the exosomes were treated with bacterial heparitinase and probed for syndecan-1 (R & D Systems).

    Techniques:

    Exosome-target cell interaction mediated by fibronectin impacts cell behavior. A , exosome interaction with RPMI-8226 cells activates p38 and ERK signaling. An antibody array that simultaneously examines the phosphorylation levels of 43 different protein kinases was utilized to determine what signaling pathways were activated when myeloma-derived exosomes interacted with target cells. RPMI-8226 cells were incubated with or without exosomes isolated from aggressive myeloma cells (CAG cells expressing high heparanase), cell lysates were exposed to membranes, and the membranes were probed with a phosphotyrosine specific antibody. Phosphorylated p38 and ERK ( circled ) were enhanced in cells incubated with the exosomes. The different phospho kinases that are activated in RPMI-8226 cells independent of the addition of exosomes are shown by arrows. Duplicate dots at the corners represent phosphotyrosine positive controls. B , RPMI-8226 cells were treated with exosomes (100 μg/ml) secreted by control or aggressive myeloma cells for 20 min and analyzed for phosphorylated p38 by Western blot. Total p38 serves as the loading control. To determine the role of cell surface heparan sulfate in mediating exosome-induced signaling, RPMI-8266 cells were treated with bacterial heparitinase for 2 h prior to the addition of exosomes from aggressive myeloma cells. C , MMP-9 and DKK1, two downstream target genes of activated p38, are up-regulated following the interaction of myeloma-derived exosomes with the RPMI 8226 cells. RPMI 8266 myeloma cells were incubated with or without exosomes, and DKK1 and MMP-9 mRNA expression in these cells was assessed using real time PCR and normalized using actin expression. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Exosome-target cell interaction mediated by fibronectin impacts cell behavior. A , exosome interaction with RPMI-8226 cells activates p38 and ERK signaling. An antibody array that simultaneously examines the phosphorylation levels of 43 different protein kinases was utilized to determine what signaling pathways were activated when myeloma-derived exosomes interacted with target cells. RPMI-8226 cells were incubated with or without exosomes isolated from aggressive myeloma cells (CAG cells expressing high heparanase), cell lysates were exposed to membranes, and the membranes were probed with a phosphotyrosine specific antibody. Phosphorylated p38 and ERK ( circled ) were enhanced in cells incubated with the exosomes. The different phospho kinases that are activated in RPMI-8226 cells independent of the addition of exosomes are shown by arrows. Duplicate dots at the corners represent phosphotyrosine positive controls. B , RPMI-8226 cells were treated with exosomes (100 μg/ml) secreted by control or aggressive myeloma cells for 20 min and analyzed for phosphorylated p38 by Western blot. Total p38 serves as the loading control. To determine the role of cell surface heparan sulfate in mediating exosome-induced signaling, RPMI-8266 cells were treated with bacterial heparitinase for 2 h prior to the addition of exosomes from aggressive myeloma cells. C , MMP-9 and DKK1, two downstream target genes of activated p38, are up-regulated following the interaction of myeloma-derived exosomes with the RPMI 8226 cells. RPMI 8266 myeloma cells were incubated with or without exosomes, and DKK1 and MMP-9 mRNA expression in these cells was assessed using real time PCR and normalized using actin expression. *, p

    Article Snippet: In some experiments, the exosomes were treated with bacterial heparitinase and probed for syndecan-1 (R & D Systems).

    Techniques: Ab Array, Derivative Assay, Incubation, Isolation, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Exosome-target cell interaction is mediated by heparan sulfate chains on target cells. A , confocal microscopy analysis of polarized or nonpolarized CAG myeloma cells following addition of CD63-RFP exosomes. Exosomes preferentially interact with heparan sulfate-rich uropods of polarized cells but are widely distributed on nonpolarized cells. B , depletion of heparan sulfate chains on myeloma cells decreases exosome-target cell interaction. RPMI-8226 cells were untreated or treated with heparitinase and washed, and their interactions with exosomes were analyzed by confocal microscopy. Right panel , quantitative data from a similar experiment analyzed by flow cytometry. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Exosome-target cell interaction is mediated by heparan sulfate chains on target cells. A , confocal microscopy analysis of polarized or nonpolarized CAG myeloma cells following addition of CD63-RFP exosomes. Exosomes preferentially interact with heparan sulfate-rich uropods of polarized cells but are widely distributed on nonpolarized cells. B , depletion of heparan sulfate chains on myeloma cells decreases exosome-target cell interaction. RPMI-8226 cells were untreated or treated with heparitinase and washed, and their interactions with exosomes were analyzed by confocal microscopy. Right panel , quantitative data from a similar experiment analyzed by flow cytometry. #, p

    Article Snippet: In some experiments, the exosomes were treated with bacterial heparitinase and probed for syndecan-1 (R & D Systems).

    Techniques: Confocal Microscopy, Flow Cytometry, Cytometry