mouse monoclonal anti-heparan sulfate antibody Search Results


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  • 94
    Thermo Fisher anti perlecan
    Double immunofluorescence staining for <t>perlecan,</t> 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections. Frozen sections (5 μm) of human atherosclerotic lesions were incubated with monoclonal or polyclonal primary antibodies: anti-nitrotyrosine (rabbit IgG), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III) anti-human CD14 (mouse mAb), non-immune rabbit IgG and non-immune mouse IgG, and subsequently the following detection antibodies: goat anti-rabbit cyanine-3 (Cy-3)-labelled IgG or goat anti-mouse Cy-2-labeled IgG. DAPI was used to image cell nuclei. Images were acquired as described in the Materials and Methods. A) Heavily thickened intima of an artery with a type III/IV lesion. Epitopes for 3-nitrotyrosine (red signal) show marked co-localization with perlecan (green) present in the basement membrane of the endothelium (arrow) as well as with those of the vasa vasorum (dotted arrow). B) The signal for 3-nitrotyrosine (red) was frequently co-localized with CD14-positive cells (macrophages, green). The red signal for nitrotyrosine underneath the bulk of macrophages results from multiple oblique sections through a vas vasorum. C) In sections where the antibodies for perlecan/CD14 and for 3-nitrotyrosine were replaced with non-immune mouse IgG (control, green) and non-immune rabbit IgG (control, red), no staining in the intima was observed. The faint background staining in the media (also observed when no primary or secondary antibodies were applied to the sections) results from elastic membranes located in this area. Scale as indicated in the bottom images.
    Anti Perlecan, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rat anti heparan sulfate proteoglycan perlecan monoclonal antibody
    Double immunofluorescence staining for <t>perlecan,</t> 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections. Frozen sections (5 μm) of human atherosclerotic lesions were incubated with monoclonal or polyclonal primary antibodies: anti-nitrotyrosine (rabbit IgG), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III) anti-human CD14 (mouse mAb), non-immune rabbit IgG and non-immune mouse IgG, and subsequently the following detection antibodies: goat anti-rabbit cyanine-3 (Cy-3)-labelled IgG or goat anti-mouse Cy-2-labeled IgG. DAPI was used to image cell nuclei. Images were acquired as described in the Materials and Methods. A) Heavily thickened intima of an artery with a type III/IV lesion. Epitopes for 3-nitrotyrosine (red signal) show marked co-localization with perlecan (green) present in the basement membrane of the endothelium (arrow) as well as with those of the vasa vasorum (dotted arrow). B) The signal for 3-nitrotyrosine (red) was frequently co-localized with CD14-positive cells (macrophages, green). The red signal for nitrotyrosine underneath the bulk of macrophages results from multiple oblique sections through a vas vasorum. C) In sections where the antibodies for perlecan/CD14 and for 3-nitrotyrosine were replaced with non-immune mouse IgG (control, green) and non-immune rabbit IgG (control, red), no staining in the intima was observed. The faint background staining in the media (also observed when no primary or secondary antibodies were applied to the sections) results from elastic membranes located in this area. Scale as indicated in the bottom images.
    Rat Anti Heparan Sulfate Proteoglycan Perlecan Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ImClone anti heparanase monoclonal antibody
    Comparison of splice 5 (S5) and wild-type (WT) human <t>heparanase</t> amino acid sequences (GCG alignment program). The two catalytic Glu residues, the proton donor and nucleophile, are marked in bold. Note that the proton donor is missing in splice 5. Potential
    Anti Heparanase Monoclonal Antibody, supplied by ImClone, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Seikagaku mouse monoclonal anti heparan sulfate antibody
    The effects of altering glucose concentrations in media for the production of serglycin, <t>heparan</t> sulfate/heparin and chondroitin sulfate. ELISA for the presence of ( A ) serglycin was detected using a polyclonal anti-serglycin antibody; ( B ) heparan sulfate/heparin stubs were detected using anti-heparan sulfate stub antibody clone 3G10 following HepIII digestion; ( C ) heparan sulfate chains were detected using anti-heparan sulfate antibody clone 10E4; and ( D ) chondroitin sulfate chains were detected using anti- chondroitin sulfate chain antibody clone CS-56. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p
    Mouse Monoclonal Anti Heparan Sulfate Antibody, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    AMS Biotechnology mouse monoclonal anti heparan sulfate
    The effects of altering glucose concentrations in media for the production of serglycin, <t>heparan</t> sulfate/heparin and chondroitin sulfate. ELISA for the presence of ( A ) serglycin was detected using a polyclonal anti-serglycin antibody; ( B ) heparan sulfate/heparin stubs were detected using anti-heparan sulfate stub antibody clone 3G10 following HepIII digestion; ( C ) heparan sulfate chains were detected using anti-heparan sulfate antibody clone 10E4; and ( D ) chondroitin sulfate chains were detected using anti- chondroitin sulfate chain antibody clone CS-56. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p
    Mouse Monoclonal Anti Heparan Sulfate, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology monoclonal mouse anti perlecan
    Immunofluorescence labeling of ECM protein digestion at the islet–exocrine interface of young and standard donors. Representative images of pancreas sections following treatment with HBSS (control), collagenase, neutral protease, or a combination of both enzymes for 5 min. Digestion of (a) collagen IV and (b) laminin-α5 (both green) is seen around and within the islet (red, insulin labeling of β-cells). Loss of signal for both proteins was significantly greater in the standard donor group than the young group when treated with neutral protease. There was no significant loss of laminin-α5 when treated with collagenase alone in either group, though this treatment was effective at digesting collagen IV in both donor groups. The loss of collagen IV by collagenase was more effective in standard donors. The combination of enzymes ameliorated the significant age-related digestion differences in both proteins. Representative images to show the effect of each enzyme component alone and in combination on digestion of (c) collagen VI (green) and (d) <t>perlecan</t> (green) both around and within the islet (red, insulin labeling of β-cells). Representative images showing the localization of each ECM protein around and within the islet. Triple immunofluorescent staining for: (e) collagen IV (green), laminin-α5 (blue), and insulin (red); (f) collagen VI (green), perlecan (blue), and insulin (red). Scale bars = 100 μm.
    Monoclonal Mouse Anti Perlecan, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology perlecan mab
    Immunofluorescence labeling of ECM protein digestion at the islet–exocrine interface of young and standard donors. Representative images of pancreas sections following treatment with HBSS (control), collagenase, neutral protease, or a combination of both enzymes for 5 min. Digestion of (a) collagen IV and (b) laminin-α5 (both green) is seen around and within the islet (red, insulin labeling of β-cells). Loss of signal for both proteins was significantly greater in the standard donor group than the young group when treated with neutral protease. There was no significant loss of laminin-α5 when treated with collagenase alone in either group, though this treatment was effective at digesting collagen IV in both donor groups. The loss of collagen IV by collagenase was more effective in standard donors. The combination of enzymes ameliorated the significant age-related digestion differences in both proteins. Representative images to show the effect of each enzyme component alone and in combination on digestion of (c) collagen VI (green) and (d) <t>perlecan</t> (green) both around and within the islet (red, insulin labeling of β-cells). Representative images showing the localization of each ECM protein around and within the islet. Triple immunofluorescent staining for: (e) collagen IV (green), laminin-α5 (blue), and insulin (red); (f) collagen VI (green), perlecan (blue), and insulin (red). Scale bars = 100 μm.
    Perlecan Mab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology mouse monoclonal anti human perlecan antibody
    EBM component mRNA expression in primary cultures of rabbit keratocytes in presence of different cytokines/growth factors. Keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 10 ng/mL IL-1β, 2 ng/mL TGF-β1, 10 ng/mL TGF-β3, 10 ng/mL PDGF-AA, or 10 ng/mL PDGF-AB for 8 or 12 hours. Expression of <t>perlecan</t> (A), nidogen-1 (B), and nidogen-2 (C) mRNA was measured by qRT-PCR and normalized to 18S rRNA as described in the material and methods section. “Co” represents primary cultured keratocan + keratocytes in the medium without added cytokines or growth factors. Data for each BM component and each cytokine or growth factor are presented as means of three independent experiments and statistical comparisons were made between vehicle-treated control keratocytes and cytokine- or growth factor–treated keratocytes at the same time points. No comparisons were made between the 8- and 12-hour time points.
    Mouse Monoclonal Anti Human Perlecan Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA rat anti mouse hspg monoclonal antibody
    EBM component mRNA expression in primary cultures of rabbit keratocytes in presence of different cytokines/growth factors. Keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 10 ng/mL IL-1β, 2 ng/mL TGF-β1, 10 ng/mL TGF-β3, 10 ng/mL PDGF-AA, or 10 ng/mL PDGF-AB for 8 or 12 hours. Expression of <t>perlecan</t> (A), nidogen-1 (B), and nidogen-2 (C) mRNA was measured by qRT-PCR and normalized to 18S rRNA as described in the material and methods section. “Co” represents primary cultured keratocan + keratocytes in the medium without added cytokines or growth factors. Data for each BM component and each cytokine or growth factor are presented as means of three independent experiments and statistical comparisons were made between vehicle-treated control keratocytes and cytokine- or growth factor–treated keratocytes at the same time points. No comparisons were made between the 8- and 12-hour time points.
    Rat Anti Mouse Hspg Monoclonal Antibody, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher rat monoclonal anti perlecan
    EBM component mRNA expression in primary cultures of rabbit keratocytes in presence of different cytokines/growth factors. Keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 10 ng/mL IL-1β, 2 ng/mL TGF-β1, 10 ng/mL TGF-β3, 10 ng/mL PDGF-AA, or 10 ng/mL PDGF-AB for 8 or 12 hours. Expression of <t>perlecan</t> (A), nidogen-1 (B), and nidogen-2 (C) mRNA was measured by qRT-PCR and normalized to 18S rRNA as described in the material and methods section. “Co” represents primary cultured keratocan + keratocytes in the medium without added cytokines or growth factors. Data for each BM component and each cytokine or growth factor are presented as means of three independent experiments and statistical comparisons were made between vehicle-treated control keratocytes and cytokine- or growth factor–treated keratocytes at the same time points. No comparisons were made between the 8- and 12-hour time points.
    Rat Monoclonal Anti Perlecan, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher optimised monoclonal mouse anti human perlecan
    Reduced basal fibronectin, type I collagen, tenascin and <t>perlecan</t> deposition upon arachidonic acid challenge. Pulmonary fibroblasts from COPD ( n = 5–6 ) and non-COPD patients ( n = 4–5 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 72 h. Deposition of fibronectin ( a ), type I collagen ( b ), tenascin ( c ) and perlecan ( d ) into the extracellular matrix (ECM) was measured by ECM ELISA. All data are expressed at fold change compared to control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p
    Optimised Monoclonal Mouse Anti Human Perlecan, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Developmental Studies Hybridoma Bank anti unc 52 perlecan mh2 monoclonal antibodies
    Reduced basal fibronectin, type I collagen, tenascin and <t>perlecan</t> deposition upon arachidonic acid challenge. Pulmonary fibroblasts from COPD ( n = 5–6 ) and non-COPD patients ( n = 4–5 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 72 h. Deposition of fibronectin ( a ), type I collagen ( b ), tenascin ( c ) and perlecan ( d ) into the extracellular matrix (ECM) was measured by ECM ELISA. All data are expressed at fold change compared to control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p
    Anti Unc 52 Perlecan Mh2 Monoclonal Antibodies, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam anti heparanase mab
    PRRSV infection upregulates <t>heparanase.</t> (A to D) Marc-145 cells or PAMs were mock infected or infected with PRRSV at an MOI of 0.1 for the indicated times. The transcript levels of heparanase were determined by qRT-PCR (A and C), and the protein levels of heparanase and PRRSV N were determined by Western blot analysis (B and D). (E) Marc-145 cells were mock infected or infected with PRRSV-EGFP at an MOI of 0.1 for the indicated times and fixed for heparanase (red) and PRRSV-EGFP (green) detection using immunofluorescence microscopy. Nucleus (blue) was stained with DAPI. Bar, 300 μm. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P
    Anti Heparanase Mab, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Eisai anti perlecan mab
    Abrogation of the <t>perlecan</t> binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.
    Anti Perlecan Mab, supplied by Eisai, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti perlecan monoclonal antibody
    Abrogation of the <t>perlecan</t> binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.
    Anti Perlecan Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Epitope Biotech anti heparan sulfate mouse mab
    Abrogation of the <t>perlecan</t> binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.
    Anti Heparan Sulfate Mouse Mab, supplied by Epitope Biotech, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher anti heparan sulfate proteoglycan perlecan
    Abrogation of the <t>perlecan</t> binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.
    Anti Heparan Sulfate Proteoglycan Perlecan, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    AMS Biotechnology anti hspg
    Abrogation of the <t>perlecan</t> binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.
    Anti Hspg, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    US Biological Life Sciences anti mouse heparan sulfate hepss 1 monoclonal antibody
    Abrogation of the <t>perlecan</t> binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.
    Anti Mouse Heparan Sulfate Hepss 1 Monoclonal Antibody, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Double immunofluorescence staining for perlecan, 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections. Frozen sections (5 μm) of human atherosclerotic lesions were incubated with monoclonal or polyclonal primary antibodies: anti-nitrotyrosine (rabbit IgG), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III) anti-human CD14 (mouse mAb), non-immune rabbit IgG and non-immune mouse IgG, and subsequently the following detection antibodies: goat anti-rabbit cyanine-3 (Cy-3)-labelled IgG or goat anti-mouse Cy-2-labeled IgG. DAPI was used to image cell nuclei. Images were acquired as described in the Materials and Methods. A) Heavily thickened intima of an artery with a type III/IV lesion. Epitopes for 3-nitrotyrosine (red signal) show marked co-localization with perlecan (green) present in the basement membrane of the endothelium (arrow) as well as with those of the vasa vasorum (dotted arrow). B) The signal for 3-nitrotyrosine (red) was frequently co-localized with CD14-positive cells (macrophages, green). The red signal for nitrotyrosine underneath the bulk of macrophages results from multiple oblique sections through a vas vasorum. C) In sections where the antibodies for perlecan/CD14 and for 3-nitrotyrosine were replaced with non-immune mouse IgG (control, green) and non-immune rabbit IgG (control, red), no staining in the intima was observed. The faint background staining in the media (also observed when no primary or secondary antibodies were applied to the sections) results from elastic membranes located in this area. Scale as indicated in the bottom images.

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Double immunofluorescence staining for perlecan, 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections. Frozen sections (5 μm) of human atherosclerotic lesions were incubated with monoclonal or polyclonal primary antibodies: anti-nitrotyrosine (rabbit IgG), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III) anti-human CD14 (mouse mAb), non-immune rabbit IgG and non-immune mouse IgG, and subsequently the following detection antibodies: goat anti-rabbit cyanine-3 (Cy-3)-labelled IgG or goat anti-mouse Cy-2-labeled IgG. DAPI was used to image cell nuclei. Images were acquired as described in the Materials and Methods. A) Heavily thickened intima of an artery with a type III/IV lesion. Epitopes for 3-nitrotyrosine (red signal) show marked co-localization with perlecan (green) present in the basement membrane of the endothelium (arrow) as well as with those of the vasa vasorum (dotted arrow). B) The signal for 3-nitrotyrosine (red) was frequently co-localized with CD14-positive cells (macrophages, green). The red signal for nitrotyrosine underneath the bulk of macrophages results from multiple oblique sections through a vas vasorum. C) In sections where the antibodies for perlecan/CD14 and for 3-nitrotyrosine were replaced with non-immune mouse IgG (control, green) and non-immune rabbit IgG (control, red), no staining in the intima was observed. The faint background staining in the media (also observed when no primary or secondary antibodies were applied to the sections) results from elastic membranes located in this area. Scale as indicated in the bottom images.

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Double Immunofluorescence Staining, Incubation, Labeling, Staining

    Effect of perlecan modification by peroxynitrite on Baf-32 cell proliferation and binding of FGF-2. (A) BaF3 cells (expressing FGF receptor 1c) were incubated with perlecan and FGF2 and the relative amount of growth was measured as described in the methods section. (B) Surface absorbed perlecan (32 nM, black bars) was exposed to peroxynitrite (ONOO, 32 μM, white bars) or dONOO (32 μM, light grey bars) in 0.1 M phosphate buffer, pH 6.5 or pH 7.5 for 20 min at 22 °C. The extent of [ 125 I]-labeled FGF-2 binding after 2 h incubation at 22 °C was quantified by gamma counting. a significantly different to untreated perlecan control.

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Effect of perlecan modification by peroxynitrite on Baf-32 cell proliferation and binding of FGF-2. (A) BaF3 cells (expressing FGF receptor 1c) were incubated with perlecan and FGF2 and the relative amount of growth was measured as described in the methods section. (B) Surface absorbed perlecan (32 nM, black bars) was exposed to peroxynitrite (ONOO, 32 μM, white bars) or dONOO (32 μM, light grey bars) in 0.1 M phosphate buffer, pH 6.5 or pH 7.5 for 20 min at 22 °C. The extent of [ 125 I]-labeled FGF-2 binding after 2 h incubation at 22 °C was quantified by gamma counting. a significantly different to untreated perlecan control.

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Modification, Binding Assay, Expressing, Incubation, Labeling

    Structural consequences of perlecan modification by peroxynitrite. Perlecan (330 nM) in 0.1 M phosphate buffer, pH 7 (lane 1) was exposed for 20 min at 22 °C to peroxynitrite at molar ratios of 250 (lane 2), 500 (lane 3), 1000 (lane 4), 1000 in the presence of 25 mM bicarbonate (lane 5), heparinase III for 16 h at 37 °C (lane 6), heparinase III followed by 1000-fold molar excess of peroxynitrite (lane 7), and dONOO (lane 8), prior to separation on 3-8% Tris-acetate gels under reducing conditions for 1 h at 150 V and subsequent western blotting to nitrocellulose. (A) Stains-all/silver stain of gel. (B) Western blot probed for 3-nitroTyr formation with mAb against 3-nitroTyr (HM11). (C) Western blot probed for heparan sulfate epitopes with mAb 10E4. (D) Western blot probed for perlecan domain III with mAb 7B5. Position of molecular mass markers are shown for reference: ◁ perlecan heparan sulfate, ◀ perlecan protein core.

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Structural consequences of perlecan modification by peroxynitrite. Perlecan (330 nM) in 0.1 M phosphate buffer, pH 7 (lane 1) was exposed for 20 min at 22 °C to peroxynitrite at molar ratios of 250 (lane 2), 500 (lane 3), 1000 (lane 4), 1000 in the presence of 25 mM bicarbonate (lane 5), heparinase III for 16 h at 37 °C (lane 6), heparinase III followed by 1000-fold molar excess of peroxynitrite (lane 7), and dONOO (lane 8), prior to separation on 3-8% Tris-acetate gels under reducing conditions for 1 h at 150 V and subsequent western blotting to nitrocellulose. (A) Stains-all/silver stain of gel. (B) Western blot probed for 3-nitroTyr formation with mAb against 3-nitroTyr (HM11). (C) Western blot probed for heparan sulfate epitopes with mAb 10E4. (D) Western blot probed for perlecan domain III with mAb 7B5. Position of molecular mass markers are shown for reference: ◁ perlecan heparan sulfate, ◀ perlecan protein core.

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Modification, Western Blot, Silver Staining

    Effect of perlecan modification by peroxynitrite on human coronary artery endothelial cell adhesion. Surface absorbed fibronectin (20 nM) and perlecan (20 nM) were exposed to peroxynitrite (ONOO, 20 μM) or dONOO (20 μM) in 0.1 M phosphate buffer, pH 6.5 or pH 7.5, in the absence and presence of NaHCO 3 (25 mM) for 20 min at 22 °C. The adhesion of endothelial cells (HCAECs) was measured by staining of bound cells with crystal violet and expressed as a percentage of adhesion to surface-adsorbed fibronectin. Data are means ± SEM of values obtained from six separate wells from a single assay, representative of three. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Effect of perlecan modification by peroxynitrite on human coronary artery endothelial cell adhesion. Surface absorbed fibronectin (20 nM) and perlecan (20 nM) were exposed to peroxynitrite (ONOO, 20 μM) or dONOO (20 μM) in 0.1 M phosphate buffer, pH 6.5 or pH 7.5, in the absence and presence of NaHCO 3 (25 mM) for 20 min at 22 °C. The adhesion of endothelial cells (HCAECs) was measured by staining of bound cells with crystal violet and expressed as a percentage of adhesion to surface-adsorbed fibronectin. Data are means ± SEM of values obtained from six separate wells from a single assay, representative of three. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Modification, Staining

    Effect of removal of the heparan sulfate (HS) chains of perlecan on modification induced by peroxynitrite. Surface absorbed perlecan (10 nM, Pln) was treated with 0.025 IU mL -1 heparinase III (HSase) for 16 h at 37 °C prior to exposure to peroxynitrite (10 μM, ONOO) in 0.1 M phosphate buffer, pH 6.5 for 20 min at 22 °C. The perlecan was then probed by ELISA using (A) mAb 10E4 (B) perlecan core (mAb CSI-076), (C) perlecan domain III (mAb 7B5) and (D) 3-nitroTyr (HM11). Data are expressed as % normal ELISA signal and are means ± SEM of values obtained from 6 wells, representative data shown. Data were analyzed by 1-way ANOVA, with statistical significance assumed at p

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Effect of removal of the heparan sulfate (HS) chains of perlecan on modification induced by peroxynitrite. Surface absorbed perlecan (10 nM, Pln) was treated with 0.025 IU mL -1 heparinase III (HSase) for 16 h at 37 °C prior to exposure to peroxynitrite (10 μM, ONOO) in 0.1 M phosphate buffer, pH 6.5 for 20 min at 22 °C. The perlecan was then probed by ELISA using (A) mAb 10E4 (B) perlecan core (mAb CSI-076), (C) perlecan domain III (mAb 7B5) and (D) 3-nitroTyr (HM11). Data are expressed as % normal ELISA signal and are means ± SEM of values obtained from 6 wells, representative data shown. Data were analyzed by 1-way ANOVA, with statistical significance assumed at p

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Modification, Enzyme-linked Immunosorbent Assay

    Computational modelling of the reaction of CO 3 •- with the protein core and heparan sulfate chains of perlecan. Modeling of the reaction of CO 3 •- with native and HS-free perlecan (10 nM) was carried out using literature abundance data and second-order rate constants (see Materials and Methods) at various molar excesses (0.1 – 10 μM) of perlecan and assuming a 35% yield of CO 3 •- . Data are presented as the predicted consumption of Tyr residues in native perlecan, expressed as a percentage of the predicted consumption of Tyr residues in HS-free perlecan, using both the literature rate constant for reaction of CO 3 •- with phenol, and a 10-fold lower value to simulate steric and electronic effects on the reaction of CO 3 •- with buried Tyr residues. In both systems, the model predicts less loss of Tyr residues in the presence of HS chains than in their absence (i.e. the HS chains act as a competitive target for CO 3 •- ), with this effect being more marked at higher oxidant excesses.

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Computational modelling of the reaction of CO 3 •- with the protein core and heparan sulfate chains of perlecan. Modeling of the reaction of CO 3 •- with native and HS-free perlecan (10 nM) was carried out using literature abundance data and second-order rate constants (see Materials and Methods) at various molar excesses (0.1 – 10 μM) of perlecan and assuming a 35% yield of CO 3 •- . Data are presented as the predicted consumption of Tyr residues in native perlecan, expressed as a percentage of the predicted consumption of Tyr residues in HS-free perlecan, using both the literature rate constant for reaction of CO 3 •- with phenol, and a 10-fold lower value to simulate steric and electronic effects on the reaction of CO 3 •- with buried Tyr residues. In both systems, the model predicts less loss of Tyr residues in the presence of HS chains than in their absence (i.e. the HS chains act as a competitive target for CO 3 •- ), with this effect being more marked at higher oxidant excesses.

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Activated Clotting Time Assay

    Effect of peroxynitrite exposure on the recognition of native epitopes on perlecan by monoclonal antibodies. Surface absorbed perlecan (10 nM) was treated with buffer (Pln), decomposed peroxynitrite (dONOO, 10 μM) or peroxynitrite (ONOO, 10 μM) at 22 °C for 20 min in 0.1 M phosphate buffer, pH 6.5 in the absence (black bars) and presence (light grey bars) of NaHCO 3 (25 mM). The perlecan was then probed by ELISA using monoclonal antibodies (mAbs) against HS epitopes: (A) mAb 10E4; (B) mAb HepSS-1; (C) mAb JM403 and protein core epitopes: (D) perlecan domain I (mAb CSI-076); (E) perlecan domain III (mAb 7B5); and (F) perlecan domain V (mAb CSI-074). Data are expressed as a percentage of the untreated proteoglycan ELISA signal, and are means ± SEM of values obtained from triplicate wells from n ≥ 3 independent experiments. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Effect of peroxynitrite exposure on the recognition of native epitopes on perlecan by monoclonal antibodies. Surface absorbed perlecan (10 nM) was treated with buffer (Pln), decomposed peroxynitrite (dONOO, 10 μM) or peroxynitrite (ONOO, 10 μM) at 22 °C for 20 min in 0.1 M phosphate buffer, pH 6.5 in the absence (black bars) and presence (light grey bars) of NaHCO 3 (25 mM). The perlecan was then probed by ELISA using monoclonal antibodies (mAbs) against HS epitopes: (A) mAb 10E4; (B) mAb HepSS-1; (C) mAb JM403 and protein core epitopes: (D) perlecan domain I (mAb CSI-076); (E) perlecan domain III (mAb 7B5); and (F) perlecan domain V (mAb CSI-074). Data are expressed as a percentage of the untreated proteoglycan ELISA signal, and are means ± SEM of values obtained from triplicate wells from n ≥ 3 independent experiments. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Enzyme-linked Immunosorbent Assay

    Effect of reaction pH on peroxynitrite-induced modifcation of perlecan. Surface absorbed perlecan (10 nM) was treated with peroxynitrite (10 μM) at 22 °C for 20 min in 0.1 M phosphate buffer, pH 6 – 7.5 in the absence (black bars) and presence (light grey bars) of NaHCO 3 (25 mM). The perlecan was then probed by ELISA using mAbs against HS epitopes; (A) mAb 10E4 and protein core epitopes; (B) perlecan core (mAb CSI-076), (C) perlecan domain III (mAb 7B5) and (D) perlecan domain V (mAb CSI-074). Data are expressed as % normal ELISA signal and are means ± SEM of values obtained from triplicate wells with n ≥ 3. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Effect of reaction pH on peroxynitrite-induced modifcation of perlecan. Surface absorbed perlecan (10 nM) was treated with peroxynitrite (10 μM) at 22 °C for 20 min in 0.1 M phosphate buffer, pH 6 – 7.5 in the absence (black bars) and presence (light grey bars) of NaHCO 3 (25 mM). The perlecan was then probed by ELISA using mAbs against HS epitopes; (A) mAb 10E4 and protein core epitopes; (B) perlecan core (mAb CSI-076), (C) perlecan domain III (mAb 7B5) and (D) perlecan domain V (mAb CSI-074). Data are expressed as % normal ELISA signal and are means ± SEM of values obtained from triplicate wells with n ≥ 3. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Enzyme-linked Immunosorbent Assay

    Formation of protein carbonyls, a generic marked of protein oxidation, on perlecan exposed to peroxynitrite. Perlecan (100 nM) was exposed to peroxynitrite (100 μM) in 0.1 M phosphate buffer, pH 7 for 20 min at 22 °C. Protein carbonyl formation was measured by ELISA.

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Formation of protein carbonyls, a generic marked of protein oxidation, on perlecan exposed to peroxynitrite. Perlecan (100 nM) was exposed to peroxynitrite (100 μM) in 0.1 M phosphate buffer, pH 7 for 20 min at 22 °C. Protein carbonyl formation was measured by ELISA.

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Enzyme-linked Immunosorbent Assay

    Formation of the tyrosine oxidation product 3-nitroTyr on perlecan exposed to peroxynitrite: (A) effect of oxidant concentration, and (B) effect of reaction pH. Surface absorbed perlecan (10 nM) was treated in the absence (black bars) and presence (light grey bars) of NaHCO 3 (25 mM) for 20 min at 22 °C with (A) 0.1 M phosphate buffer, pH 7.5 containing 1 – 10 μM peroxynitrite (100 – 1000 molar excess of oxidant over protein) or dONOO (10 μM) or (B) 0.1 M phosphate buffer, pH 6 – 7.5 containing 10 μM peroxynitrite. The perlecan was then probed by ELISA using a mAb against 3-nitroTyr (HM11). Data are expressed as a % of the control ELISA signal obtained with untreated perlecan and are means ± SEM of values obtained from triplicate wells with n ≥ 3. The absolute absorbance (at 405 nm) for the controls with no perlecan were ∼ 0.055, and for native perlecan ∼ 0.09; those for the peroxynitrite-treated perlecan were up to 1.6 absorbance units. These data indicate that the level of 3-nitroTyr on the native perlecan was very low. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Journal: Free Radical Biology & Medicine

    Article Title: Peroxynitrite modifies the structure and function of the extracellular matrix proteoglycan perlecan by reaction with both the protein core and the heparan sulfate chains

    doi: 10.1016/j.freeradbiomed.2010.04.018

    Figure Lengend Snippet: Formation of the tyrosine oxidation product 3-nitroTyr on perlecan exposed to peroxynitrite: (A) effect of oxidant concentration, and (B) effect of reaction pH. Surface absorbed perlecan (10 nM) was treated in the absence (black bars) and presence (light grey bars) of NaHCO 3 (25 mM) for 20 min at 22 °C with (A) 0.1 M phosphate buffer, pH 7.5 containing 1 – 10 μM peroxynitrite (100 – 1000 molar excess of oxidant over protein) or dONOO (10 μM) or (B) 0.1 M phosphate buffer, pH 6 – 7.5 containing 10 μM peroxynitrite. The perlecan was then probed by ELISA using a mAb against 3-nitroTyr (HM11). Data are expressed as a % of the control ELISA signal obtained with untreated perlecan and are means ± SEM of values obtained from triplicate wells with n ≥ 3. The absolute absorbance (at 405 nm) for the controls with no perlecan were ∼ 0.055, and for native perlecan ∼ 0.09; those for the peroxynitrite-treated perlecan were up to 1.6 absorbance units. These data indicate that the level of 3-nitroTyr on the native perlecan was very low. Data were analyzed by 2-way ANOVA, with statistical significance assumed at p

    Article Snippet: The following monoclonal or polyclonal antibodies were used as primary antibodies: anti-nitrotyrosine (rabbit IgG, Millipore, 1:75 dilution), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III, Zymed Laboratories, 200 µg/ml, 1:10 dilution) anti-human CD14 (mouse mAb recognizing monocytes/macrophages, Serotec, 1:50 dilution), non-immune rabbit IgG (Sigma) and non-immune mouse IgG (Sigma).

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Effects of basement membranes and perlecan on serum-stimulated MAPK activation. (A) SMCs were replated on basement membranes (BM), FN, LN, or IV, or Pl in the presence (black bars) or absence (white bars) of 10% NCS for 4 h. Total MAPK (ERK 1 and 2) activity was measured as described in MATERIALS AND METHODS. The data are shown as picomoles ATP utilized per minute per milligram total protein and are presented as the mean ± SEM of three separate experiments. (B) SMCs were replated on PN or FN matrices in the presence of 10% CS. Whole-cell lysates were collected 4 h after replating, and equal protein concentrations were analyzed by Western analysis for phosphorylated ERK1/2 (top). The filter was stripped and reprobed with a total ERK1/2 antibody (bottom). Shown in the Western blot are two independent experiments for each matrix protein.

    Journal: Molecular Biology of the Cell

    Article Title: Perlecan Up-Regulation of FRNK Suppresses Smooth Muscle Cell Proliferation via Inhibition of FAK Signaling

    doi: 10.1091/mbc.E02-08-0508

    Figure Lengend Snippet: Effects of basement membranes and perlecan on serum-stimulated MAPK activation. (A) SMCs were replated on basement membranes (BM), FN, LN, or IV, or Pl in the presence (black bars) or absence (white bars) of 10% NCS for 4 h. Total MAPK (ERK 1 and 2) activity was measured as described in MATERIALS AND METHODS. The data are shown as picomoles ATP utilized per minute per milligram total protein and are presented as the mean ± SEM of three separate experiments. (B) SMCs were replated on PN or FN matrices in the presence of 10% CS. Whole-cell lysates were collected 4 h after replating, and equal protein concentrations were analyzed by Western analysis for phosphorylated ERK1/2 (top). The filter was stripped and reprobed with a total ERK1/2 antibody (bottom). Shown in the Western blot are two independent experiments for each matrix protein.

    Article Snippet: Monoclonal anti-perlecan antibody was from Zymed Laboratories (South San Francisco, CA).

    Techniques: Activation Assay, Activity Assay, Western Blot

    Up-regulation of FRNK by perlecan is actively and continuously regulated. (A) A10 SMCs were transfected with a pXP2-FRNK-LUC construct or a promoterless pXP2-LUC construct, and cell lysates were assayed for luciferase activity as described in MATERIALS AND METHODS (1 μg of test DNA plus 0.5 μg of CMV-βGal per transfection). β-Galactosidase activity of each lysate was measured, and luciferase activity for each lysate was normalized to the relative β-galactosidase activity of that lysate to give relative light units. Data are reported as fold activation ± SEM over promoter-less pXP2 vector (control set to 1) of three separate transfections. *p

    Journal: Molecular Biology of the Cell

    Article Title: Perlecan Up-Regulation of FRNK Suppresses Smooth Muscle Cell Proliferation via Inhibition of FAK Signaling

    doi: 10.1091/mbc.E02-08-0508

    Figure Lengend Snippet: Up-regulation of FRNK by perlecan is actively and continuously regulated. (A) A10 SMCs were transfected with a pXP2-FRNK-LUC construct or a promoterless pXP2-LUC construct, and cell lysates were assayed for luciferase activity as described in MATERIALS AND METHODS (1 μg of test DNA plus 0.5 μg of CMV-βGal per transfection). β-Galactosidase activity of each lysate was measured, and luciferase activity for each lysate was normalized to the relative β-galactosidase activity of that lysate to give relative light units. Data are reported as fold activation ± SEM over promoter-less pXP2 vector (control set to 1) of three separate transfections. *p

    Article Snippet: Monoclonal anti-perlecan antibody was from Zymed Laboratories (South San Francisco, CA).

    Techniques: Transfection, Construct, Luciferase, Activity Assay, Activation Assay, Plasmid Preparation

    Perlecan suppresses FAK activation and modulates actin microfilament formation. (A) Medial SMCs were isolated from uninjured rat aortas, and total protein was extracted (in vivo). Subcultured SMCs were left in suspension (suspended) in 10% CS or were plated on Pl, IV, FN, LN, PN, or basement membranes (BM) in 10% CS. Cell lysates were prepared after 4 h, and equal protein concentrations were analyzed by Western blotting. Shown is a representative Western blot. Top, FAK phosphorylation levels using an anti–phosphoY 397 -FAK antibody. Bottom, total FAK protein as well as equal protein loading as determined by anti-FAK immunoblotting. Western blots were scored for relative densitometry, and the data are presented in the graph as the mean ± SEM of at least three independent experiments. (B) SMCs were plated on PN or FN matrices in the presence of 10% CS and allowed to attach for 4 h. SMCs were immunofluorescently stained with rhodamine-labeled phalloidin to detect F-actin stress fiber formation and with DAPI to identify total cells.

    Journal: Molecular Biology of the Cell

    Article Title: Perlecan Up-Regulation of FRNK Suppresses Smooth Muscle Cell Proliferation via Inhibition of FAK Signaling

    doi: 10.1091/mbc.E02-08-0508

    Figure Lengend Snippet: Perlecan suppresses FAK activation and modulates actin microfilament formation. (A) Medial SMCs were isolated from uninjured rat aortas, and total protein was extracted (in vivo). Subcultured SMCs were left in suspension (suspended) in 10% CS or were plated on Pl, IV, FN, LN, PN, or basement membranes (BM) in 10% CS. Cell lysates were prepared after 4 h, and equal protein concentrations were analyzed by Western blotting. Shown is a representative Western blot. Top, FAK phosphorylation levels using an anti–phosphoY 397 -FAK antibody. Bottom, total FAK protein as well as equal protein loading as determined by anti-FAK immunoblotting. Western blots were scored for relative densitometry, and the data are presented in the graph as the mean ± SEM of at least three independent experiments. (B) SMCs were plated on PN or FN matrices in the presence of 10% CS and allowed to attach for 4 h. SMCs were immunofluorescently stained with rhodamine-labeled phalloidin to detect F-actin stress fiber formation and with DAPI to identify total cells.

    Article Snippet: Monoclonal anti-perlecan antibody was from Zymed Laboratories (South San Francisco, CA).

    Techniques: Activation Assay, Isolation, In Vivo, Western Blot, Staining, Labeling

    Effects of constitutively active FAK on perlecan-mediated SMC growth suppression. A10 SMCs were transiently transfected with constitutively active, wild-type IL2R-FAK (wt) or with dominant negative, kinase-inactivated IL2R-FAK Y397F , allowed to recover for 24 h, then were replated on coverslips precoated with (A) PN or (B) FN in the presence of 10% CS and 10 mM BrdU. Cells were fixed 24 h later and immunofluorescently stained for BrdU and IL2R. Cells were counterstained with DAPI to identify total SMCs. The percentage of BrdU-positive SMCs was determined independently for nontransfected (IL2R-negative) and transfected (IL2R-positive) SMCs by counting a minimum of 200 SMCs per condition per experiment. The data are presented as the mean ± SEM. *p

    Journal: Molecular Biology of the Cell

    Article Title: Perlecan Up-Regulation of FRNK Suppresses Smooth Muscle Cell Proliferation via Inhibition of FAK Signaling

    doi: 10.1091/mbc.E02-08-0508

    Figure Lengend Snippet: Effects of constitutively active FAK on perlecan-mediated SMC growth suppression. A10 SMCs were transiently transfected with constitutively active, wild-type IL2R-FAK (wt) or with dominant negative, kinase-inactivated IL2R-FAK Y397F , allowed to recover for 24 h, then were replated on coverslips precoated with (A) PN or (B) FN in the presence of 10% CS and 10 mM BrdU. Cells were fixed 24 h later and immunofluorescently stained for BrdU and IL2R. Cells were counterstained with DAPI to identify total SMCs. The percentage of BrdU-positive SMCs was determined independently for nontransfected (IL2R-negative) and transfected (IL2R-positive) SMCs by counting a minimum of 200 SMCs per condition per experiment. The data are presented as the mean ± SEM. *p

    Article Snippet: Monoclonal anti-perlecan antibody was from Zymed Laboratories (South San Francisco, CA).

    Techniques: Transfection, Dominant Negative Mutation, Staining

    Suppression of serum-induced SMC growth by perlecan. (A) SMCs were plated on Pl, FN, LN, IV, or PN. Some SMCs plated on Pl were treated with 100 μg/ml HS or 10 μg/ml CSPG. SMCs were growth-arrested for 72 h in SFM, then stimulated with 10% CS (black bars) or kept in SFM (white bars) for an additional 24 h in the presence of 10 mM BrdU. SMCs were fixed and immunocytochemically stained for BrdU. The percentage of BrdU-positive SMCs was determined for each condition. A minimum of 500 cells per condition was counted, and the data are presented as the mean ± SEM. (B) SMCs were plated on Pl and allowed to attach overnight. Total cell numbers were determined in triplicate after 3 and 5 d of serum stimulation plus or minus antibodies. White bars, no IgG; black bars, plus 10 μg/ml neutralizing anti-perlecan antibody; gray bars, plus nonspecific IgG. The data are presented as the mean ± SEM. *p

    Journal: Molecular Biology of the Cell

    Article Title: Perlecan Up-Regulation of FRNK Suppresses Smooth Muscle Cell Proliferation via Inhibition of FAK Signaling

    doi: 10.1091/mbc.E02-08-0508

    Figure Lengend Snippet: Suppression of serum-induced SMC growth by perlecan. (A) SMCs were plated on Pl, FN, LN, IV, or PN. Some SMCs plated on Pl were treated with 100 μg/ml HS or 10 μg/ml CSPG. SMCs were growth-arrested for 72 h in SFM, then stimulated with 10% CS (black bars) or kept in SFM (white bars) for an additional 24 h in the presence of 10 mM BrdU. SMCs were fixed and immunocytochemically stained for BrdU. The percentage of BrdU-positive SMCs was determined for each condition. A minimum of 500 cells per condition was counted, and the data are presented as the mean ± SEM. (B) SMCs were plated on Pl and allowed to attach overnight. Total cell numbers were determined in triplicate after 3 and 5 d of serum stimulation plus or minus antibodies. White bars, no IgG; black bars, plus 10 μg/ml neutralizing anti-perlecan antibody; gray bars, plus nonspecific IgG. The data are presented as the mean ± SEM. *p

    Article Snippet: Monoclonal anti-perlecan antibody was from Zymed Laboratories (South San Francisco, CA).

    Techniques: Staining

    Comparison of splice 5 (S5) and wild-type (WT) human heparanase amino acid sequences (GCG alignment program). The two catalytic Glu residues, the proton donor and nucleophile, are marked in bold. Note that the proton donor is missing in splice 5. Potential

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Comparison of splice 5 (S5) and wild-type (WT) human heparanase amino acid sequences (GCG alignment program). The two catalytic Glu residues, the proton donor and nucleophile, are marked in bold. Note that the proton donor is missing in splice 5. Potential

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques:

    Nucleotide and predicted amino acid sequences of splice 5 human heparanase. Nucleotide sequences are shown above the predicted amino acid sequences. Numbers on the left correspond to nucleotides (Roman) and amino acid residues (bold italic). The signal

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Nucleotide and predicted amino acid sequences of splice 5 human heparanase. Nucleotide sequences are shown above the predicted amino acid sequences. Numbers on the left correspond to nucleotides (Roman) and amino acid residues (bold italic). The signal

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques:

    Cloning and expression of human heparanase splice variant lacking exon 5. A. Semi-quantitive RT-PCR using primers located around the human heparanase cDNA region encoded by exon 5. Bands of 579 bp represent the wild type enzyme, while those of 405 bp

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Cloning and expression of human heparanase splice variant lacking exon 5. A. Semi-quantitive RT-PCR using primers located around the human heparanase cDNA region encoded by exon 5. Bands of 579 bp represent the wild type enzyme, while those of 405 bp

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques: Clone Assay, Expressing, Variant Assay, Reverse Transcription Polymerase Chain Reaction

    Localization of Human Heparanase and its splice variant in glioma cells. U87 cells stably transfected with the full length wild type (WT) human heparanase or its splice variant #5 (S5), were immunostained with monoclonal anti-heparanase antibody. Note

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Localization of Human Heparanase and its splice variant in glioma cells. U87 cells stably transfected with the full length wild type (WT) human heparanase or its splice variant #5 (S5), were immunostained with monoclonal anti-heparanase antibody. Note

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques: Variant Assay, Stable Transfection, Transfection

    The effects of altering glucose concentrations in media for the production of serglycin, heparan sulfate/heparin and chondroitin sulfate. ELISA for the presence of ( A ) serglycin was detected using a polyclonal anti-serglycin antibody; ( B ) heparan sulfate/heparin stubs were detected using anti-heparan sulfate stub antibody clone 3G10 following HepIII digestion; ( C ) heparan sulfate chains were detected using anti-heparan sulfate antibody clone 10E4; and ( D ) chondroitin sulfate chains were detected using anti- chondroitin sulfate chain antibody clone CS-56. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

    doi: 10.3390/molecules22050806

    Figure Lengend Snippet: The effects of altering glucose concentrations in media for the production of serglycin, heparan sulfate/heparin and chondroitin sulfate. ELISA for the presence of ( A ) serglycin was detected using a polyclonal anti-serglycin antibody; ( B ) heparan sulfate/heparin stubs were detected using anti-heparan sulfate stub antibody clone 3G10 following HepIII digestion; ( C ) heparan sulfate chains were detected using anti-heparan sulfate antibody clone 10E4; and ( D ) chondroitin sulfate chains were detected using anti- chondroitin sulfate chain antibody clone CS-56. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p

    Article Snippet: Primary antibodies used included a rabbit polyclonal anti-serglycin antibody (ascites 1:5000), mouse monoclonal anti-4-sulfated chondroitin sulfate stub antibody (clone 2B6, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-6-sulfated chondroitin sulfate stub antibody (clone 3B3, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-heparan sulfate stub antibody (clone 3G10, Seikagaku, 1 µg/mL), mouse monoclonal anti-chondroitin sulfate type A and C antibody (clone CS-56, ascites 1:2500), and mouse monoclonal anti-heparan sulfate antibody (clone 10E4, Seikagaku Corp., 1 µg/mL).

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effect of bioreactors on heparan and chondroitin sulfate structure. ELISA for the presence of ( A ) heparan sulfate chains detected using anti-heparan sulfate chain antibody clone 10E4 and ( B ) chondroitin sulfate chains detected using anti-chondroitin sulfate chain antibody clone CS-56. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

    doi: 10.3390/molecules22050806

    Figure Lengend Snippet: Effect of bioreactors on heparan and chondroitin sulfate structure. ELISA for the presence of ( A ) heparan sulfate chains detected using anti-heparan sulfate chain antibody clone 10E4 and ( B ) chondroitin sulfate chains detected using anti-chondroitin sulfate chain antibody clone CS-56. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p

    Article Snippet: Primary antibodies used included a rabbit polyclonal anti-serglycin antibody (ascites 1:5000), mouse monoclonal anti-4-sulfated chondroitin sulfate stub antibody (clone 2B6, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-6-sulfated chondroitin sulfate stub antibody (clone 3B3, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-heparan sulfate stub antibody (clone 3G10, Seikagaku, 1 µg/mL), mouse monoclonal anti-chondroitin sulfate type A and C antibody (clone CS-56, ascites 1:2500), and mouse monoclonal anti-heparan sulfate antibody (clone 10E4, Seikagaku Corp., 1 µg/mL).

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Activity of serglycin with heparin/heparan sulfate and chondroitin sulfate chains determined by the signaling of FGF receptor type 1c expressing BaF32 cells in the presence of FGF-2 as mesured by the MTS assay. Cells in the presence of FGF-2 and heparin were used as a control for the formation of active ternary complexes. Negative controls were cells in the presence of no additives, heparin, or FGF-2. Selected serglycin preparations were digested with either chondroitinase ABC, hepIII or both glycosaminoglycan lysases prior to the assay. Cell proliferation was measured after 72 h. * indicated significant differences ( p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

    doi: 10.3390/molecules22050806

    Figure Lengend Snippet: Activity of serglycin with heparin/heparan sulfate and chondroitin sulfate chains determined by the signaling of FGF receptor type 1c expressing BaF32 cells in the presence of FGF-2 as mesured by the MTS assay. Cells in the presence of FGF-2 and heparin were used as a control for the formation of active ternary complexes. Negative controls were cells in the presence of no additives, heparin, or FGF-2. Selected serglycin preparations were digested with either chondroitinase ABC, hepIII or both glycosaminoglycan lysases prior to the assay. Cell proliferation was measured after 72 h. * indicated significant differences ( p

    Article Snippet: Primary antibodies used included a rabbit polyclonal anti-serglycin antibody (ascites 1:5000), mouse monoclonal anti-4-sulfated chondroitin sulfate stub antibody (clone 2B6, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-6-sulfated chondroitin sulfate stub antibody (clone 3B3, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-heparan sulfate stub antibody (clone 3G10, Seikagaku, 1 µg/mL), mouse monoclonal anti-chondroitin sulfate type A and C antibody (clone CS-56, ascites 1:2500), and mouse monoclonal anti-heparan sulfate antibody (clone 10E4, Seikagaku Corp., 1 µg/mL).

    Techniques: Activity Assay, Expressing, MTS Assay

    The effect of bioreactors on the production of serglycin, heparin/heparan sulfate and chondroitin sulfate. The schematic indicates the structure of serglycin with eight glycosaminoglycan attachment sites that can be decorated with either chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The effect of glycosaminoglycan lyase digestion on the glycosaminoglycan chains are indicated in panels ( B , C ) with HepIII removing heparin/heparan sulfate chains to reveal a single stub structure and chondroitinase ABC (C’ase ABC) removing chondroitin/dermatan sulfate chains to reveal a stub structure. ELISA for the presence of ( A ) serglycin core protein; ( B ) heparin/heparan sulfate stubs detected using anti-heparan sulfate/heparin-stub antibody clone 3G10 following HepIII digestion, and ( C ) chondroitin sulfate stubs detected using anti-4-sulfated chondroitin sulfate stub antibody clone 2B6 and anti-6-sulfated chondroitin sulfate stub antibody clone 3B3 following C’ase ABC digestion. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

    doi: 10.3390/molecules22050806

    Figure Lengend Snippet: The effect of bioreactors on the production of serglycin, heparin/heparan sulfate and chondroitin sulfate. The schematic indicates the structure of serglycin with eight glycosaminoglycan attachment sites that can be decorated with either chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The effect of glycosaminoglycan lyase digestion on the glycosaminoglycan chains are indicated in panels ( B , C ) with HepIII removing heparin/heparan sulfate chains to reveal a single stub structure and chondroitinase ABC (C’ase ABC) removing chondroitin/dermatan sulfate chains to reveal a stub structure. ELISA for the presence of ( A ) serglycin core protein; ( B ) heparin/heparan sulfate stubs detected using anti-heparan sulfate/heparin-stub antibody clone 3G10 following HepIII digestion, and ( C ) chondroitin sulfate stubs detected using anti-4-sulfated chondroitin sulfate stub antibody clone 2B6 and anti-6-sulfated chondroitin sulfate stub antibody clone 3B3 following C’ase ABC digestion. Data are presented as means ± standard deviation ( n = 3). * indicates significant differences ( p

    Article Snippet: Primary antibodies used included a rabbit polyclonal anti-serglycin antibody (ascites 1:5000), mouse monoclonal anti-4-sulfated chondroitin sulfate stub antibody (clone 2B6, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-6-sulfated chondroitin sulfate stub antibody (clone 3B3, gift from Prof Bruce Caterson, Cardiff University, conditioned medium 1:1000), mouse monoclonal anti-heparan sulfate stub antibody (clone 3G10, Seikagaku, 1 µg/mL), mouse monoclonal anti-chondroitin sulfate type A and C antibody (clone CS-56, ascites 1:2500), and mouse monoclonal anti-heparan sulfate antibody (clone 10E4, Seikagaku Corp., 1 µg/mL).

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Immunofluorescence labeling of ECM protein digestion at the islet–exocrine interface of young and standard donors. Representative images of pancreas sections following treatment with HBSS (control), collagenase, neutral protease, or a combination of both enzymes for 5 min. Digestion of (a) collagen IV and (b) laminin-α5 (both green) is seen around and within the islet (red, insulin labeling of β-cells). Loss of signal for both proteins was significantly greater in the standard donor group than the young group when treated with neutral protease. There was no significant loss of laminin-α5 when treated with collagenase alone in either group, though this treatment was effective at digesting collagen IV in both donor groups. The loss of collagen IV by collagenase was more effective in standard donors. The combination of enzymes ameliorated the significant age-related digestion differences in both proteins. Representative images to show the effect of each enzyme component alone and in combination on digestion of (c) collagen VI (green) and (d) perlecan (green) both around and within the islet (red, insulin labeling of β-cells). Representative images showing the localization of each ECM protein around and within the islet. Triple immunofluorescent staining for: (e) collagen IV (green), laminin-α5 (blue), and insulin (red); (f) collagen VI (green), perlecan (blue), and insulin (red). Scale bars = 100 μm.

    Journal: Cell Transplantation

    Article Title: Development of a Simple In Vitro Assay to Assess Digestion of the Extracellular Matrix of the Human Pancreas by Collagenase Enzyme Blends

    doi: 10.1177/0963689718779778

    Figure Lengend Snippet: Immunofluorescence labeling of ECM protein digestion at the islet–exocrine interface of young and standard donors. Representative images of pancreas sections following treatment with HBSS (control), collagenase, neutral protease, or a combination of both enzymes for 5 min. Digestion of (a) collagen IV and (b) laminin-α5 (both green) is seen around and within the islet (red, insulin labeling of β-cells). Loss of signal for both proteins was significantly greater in the standard donor group than the young group when treated with neutral protease. There was no significant loss of laminin-α5 when treated with collagenase alone in either group, though this treatment was effective at digesting collagen IV in both donor groups. The loss of collagen IV by collagenase was more effective in standard donors. The combination of enzymes ameliorated the significant age-related digestion differences in both proteins. Representative images to show the effect of each enzyme component alone and in combination on digestion of (c) collagen VI (green) and (d) perlecan (green) both around and within the islet (red, insulin labeling of β-cells). Representative images showing the localization of each ECM protein around and within the islet. Triple immunofluorescent staining for: (e) collagen IV (green), laminin-α5 (blue), and insulin (red); (f) collagen VI (green), perlecan (blue), and insulin (red). Scale bars = 100 μm.

    Article Snippet: For sections not undergoing enzyme treatment, slides were treated as above but without the initial digestion step – that is, sections were thawed, fixed, and blocked, then incubated with primary antibodies as above for collagen IV and laminin- α5, or with polyclonal goat anti-collagen VI (1:50; BioRad) and monoclonal mouse anti-perlecan (1:100; Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Immunofluorescence, Labeling, Staining

    EBM component mRNA expression in primary cultures of rabbit keratocytes in presence of different cytokines/growth factors. Keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 10 ng/mL IL-1β, 2 ng/mL TGF-β1, 10 ng/mL TGF-β3, 10 ng/mL PDGF-AA, or 10 ng/mL PDGF-AB for 8 or 12 hours. Expression of perlecan (A), nidogen-1 (B), and nidogen-2 (C) mRNA was measured by qRT-PCR and normalized to 18S rRNA as described in the material and methods section. “Co” represents primary cultured keratocan + keratocytes in the medium without added cytokines or growth factors. Data for each BM component and each cytokine or growth factor are presented as means of three independent experiments and statistical comparisons were made between vehicle-treated control keratocytes and cytokine- or growth factor–treated keratocytes at the same time points. No comparisons were made between the 8- and 12-hour time points.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: IL-1 and TGF-β Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells

    doi: 10.1167/iovs.18-25202

    Figure Lengend Snippet: EBM component mRNA expression in primary cultures of rabbit keratocytes in presence of different cytokines/growth factors. Keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 10 ng/mL IL-1β, 2 ng/mL TGF-β1, 10 ng/mL TGF-β3, 10 ng/mL PDGF-AA, or 10 ng/mL PDGF-AB for 8 or 12 hours. Expression of perlecan (A), nidogen-1 (B), and nidogen-2 (C) mRNA was measured by qRT-PCR and normalized to 18S rRNA as described in the material and methods section. “Co” represents primary cultured keratocan + keratocytes in the medium without added cytokines or growth factors. Data for each BM component and each cytokine or growth factor are presented as means of three independent experiments and statistical comparisons were made between vehicle-treated control keratocytes and cytokine- or growth factor–treated keratocytes at the same time points. No comparisons were made between the 8- and 12-hour time points.

    Article Snippet: IHC for perlecan was performed using a mouse monoclonal anti-human perlecan antibody (Clone E6; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) that was previously shown to work in IHC against rabbit antigen at 1:50 dilution in 1% BSA in PBS for 1 hour at room temperature.

    Techniques: Expressing, Cell Culture, Quantitative RT-PCR

    Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: IL-1 and TGF-β Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells

    doi: 10.1167/iovs.18-25202

    Figure Lengend Snippet: Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Article Snippet: IHC for perlecan was performed using a mouse monoclonal anti-human perlecan antibody (Clone E6; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) that was previously shown to work in IHC against rabbit antigen at 1:50 dilution in 1% BSA in PBS for 1 hour at room temperature.

    Techniques: Expressing, Cell Culture, Western Blot

    Immunohistochemistry for perlecan protein expression in control unwounded corneas and at time points after −4.5- and −9-D PRK in rabbits. e is epithelium and S is stroma in each panel. Blue is DAPI staining of all nuclei in all panels. In each case, the panel shown is representative of the results noted in three corneas at each time point in each group. (A) Example control staining (this example at 2 days after −9-D PRK) with no primary antibody. (B) In unwounded control corneas perlecan was detected in the EBM (arrowheads) but little was detected in stromal cells. At (C) 1 day after −9-D PRK (D) 1 day after −4.5-D PRK, perlecan protein production was present in some stromal cells (arrows). At (E) 2 days after −9-D PRK, (F) 2 days after −4.5-D PRK, (G) 4 days after −9-D PRK, or (H) 4 days after −4.5-D PRK there was similar perlecan in the nascent EBM (arrowheads) and stromal cells (arrows). At 7 days after −9-D PRK, a change was noted in subepithelial EBM perlecan (arrowheads) in the (I) −9-D PRK corneas compared with the (J) −4.5-D PRK corneas. At 14 days, after the difference in the subepithelial perlecan (arrowheads) between (K) −9-D and (L) −4.5-D PRK was even more pronounced. Perlecan was detected in anterior stromal cells in both groups (arrows). At (M) 30 days after −9-D PRK there continued to be no subepithelial linear EBM perlecan, although perlecan was detected in cells in the anterior stroma (arrows). In contrast, in corneas (N) at 30 days after −4.5-D PRK, the linear EBM-associated perlecan (arrowheads) was present and there was relatively little perlecan present in stromal cells. Magnification ×200 in all panels.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: IL-1 and TGF-β Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells

    doi: 10.1167/iovs.18-25202

    Figure Lengend Snippet: Immunohistochemistry for perlecan protein expression in control unwounded corneas and at time points after −4.5- and −9-D PRK in rabbits. e is epithelium and S is stroma in each panel. Blue is DAPI staining of all nuclei in all panels. In each case, the panel shown is representative of the results noted in three corneas at each time point in each group. (A) Example control staining (this example at 2 days after −9-D PRK) with no primary antibody. (B) In unwounded control corneas perlecan was detected in the EBM (arrowheads) but little was detected in stromal cells. At (C) 1 day after −9-D PRK (D) 1 day after −4.5-D PRK, perlecan protein production was present in some stromal cells (arrows). At (E) 2 days after −9-D PRK, (F) 2 days after −4.5-D PRK, (G) 4 days after −9-D PRK, or (H) 4 days after −4.5-D PRK there was similar perlecan in the nascent EBM (arrowheads) and stromal cells (arrows). At 7 days after −9-D PRK, a change was noted in subepithelial EBM perlecan (arrowheads) in the (I) −9-D PRK corneas compared with the (J) −4.5-D PRK corneas. At 14 days, after the difference in the subepithelial perlecan (arrowheads) between (K) −9-D and (L) −4.5-D PRK was even more pronounced. Perlecan was detected in anterior stromal cells in both groups (arrows). At (M) 30 days after −9-D PRK there continued to be no subepithelial linear EBM perlecan, although perlecan was detected in cells in the anterior stroma (arrows). In contrast, in corneas (N) at 30 days after −4.5-D PRK, the linear EBM-associated perlecan (arrowheads) was present and there was relatively little perlecan present in stromal cells. Magnification ×200 in all panels.

    Article Snippet: IHC for perlecan was performed using a mouse monoclonal anti-human perlecan antibody (Clone E6; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) that was previously shown to work in IHC against rabbit antigen at 1:50 dilution in 1% BSA in PBS for 1 hour at room temperature.

    Techniques: Immunohistochemistry, Expressing, Staining

    Reduced basal fibronectin, type I collagen, tenascin and perlecan deposition upon arachidonic acid challenge. Pulmonary fibroblasts from COPD ( n = 5–6 ) and non-COPD patients ( n = 4–5 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 72 h. Deposition of fibronectin ( a ), type I collagen ( b ), tenascin ( c ) and perlecan ( d ) into the extracellular matrix (ECM) was measured by ECM ELISA. All data are expressed at fold change compared to control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p

    Journal: Respiratory Research

    Article Title: Dietary ω-6 polyunsaturated fatty acid arachidonic acid increases inflammation, but inhibits ECM protein expression in COPD

    doi: 10.1186/s12931-018-0919-4

    Figure Lengend Snippet: Reduced basal fibronectin, type I collagen, tenascin and perlecan deposition upon arachidonic acid challenge. Pulmonary fibroblasts from COPD ( n = 5–6 ) and non-COPD patients ( n = 4–5 ) were unstimulated (control) or challenged with the ω-6 PUFA arachidonic acid (AA) in 0.1% BSA-DMEM (100 μM) for 72 h. Deposition of fibronectin ( a ), type I collagen ( b ), tenascin ( c ) and perlecan ( d ) into the extracellular matrix (ECM) was measured by ECM ELISA. All data are expressed at fold change compared to control ± standard error of the mean. Challenge with AA is compared to control using a Two-way ANOVA with LSD fisher’s test. Significance is represented as * ( p

    Article Snippet: Measurement of ECM protein deposition Fibronectin, type I collagen, tenascin and perlecan deposition into the ECM was measured by ECM ELISA using optimised monoclonal mouse-anti human perlecan (2 μg/ml) (ThermoFisher Scientific), type I collagen (2 μg/ml)(Sigma-Aldrich), fibronectin (0.5 μg/ml) (Merck, Bayswater, Australia) and tenascin (0.5 μg/ml) (Sigma-Aldrich) antibodies as previously described by Kuo et al. (2011) [ ].

    Techniques: Enzyme-linked Immunosorbent Assay

    PRRSV infection upregulates heparanase. (A to D) Marc-145 cells or PAMs were mock infected or infected with PRRSV at an MOI of 0.1 for the indicated times. The transcript levels of heparanase were determined by qRT-PCR (A and C), and the protein levels of heparanase and PRRSV N were determined by Western blot analysis (B and D). (E) Marc-145 cells were mock infected or infected with PRRSV-EGFP at an MOI of 0.1 for the indicated times and fixed for heparanase (red) and PRRSV-EGFP (green) detection using immunofluorescence microscopy. Nucleus (blue) was stained with DAPI. Bar, 300 μm. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Journal: Journal of Virology

    Article Title: Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

    doi: 10.1128/JVI.00625-17

    Figure Lengend Snippet: PRRSV infection upregulates heparanase. (A to D) Marc-145 cells or PAMs were mock infected or infected with PRRSV at an MOI of 0.1 for the indicated times. The transcript levels of heparanase were determined by qRT-PCR (A and C), and the protein levels of heparanase and PRRSV N were determined by Western blot analysis (B and D). (E) Marc-145 cells were mock infected or infected with PRRSV-EGFP at an MOI of 0.1 for the indicated times and fixed for heparanase (red) and PRRSV-EGFP (green) detection using immunofluorescence microscopy. Nucleus (blue) was stained with DAPI. Bar, 300 μm. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Article Snippet: For Western blot experiments, the primary antibodies anti-PRRSV N protein MAb (1:2,000; Jeno Biotech, Inc., Republic of Korea), anti-heparanase MAb (1:1,000; Abcam), anti-NF-κB p-IκBα MAb (1:2,000; Cell Signal Technology), anti-cathepsin L (1:1,000; Abcam), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) MAb (1:2,000; Cell Signal Technology), horseradish peroxidase-conjugated anti-mouse IgG antibody, and anti-rabbit IgG antibody (1:2,000; Cell Signal Technology) were used.

    Techniques: Infection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Microscopy, Staining

    Mechanism of heparanase upregulation after PRRSV infection. (A) Marc-145 cells were inoculated with PRRSV (MOI of 0.1) and then harvested for qRT-PCR analysis of NF-κB p65 expression at 6, 12, and 24 hpi. (B) Cells were mock infected or infected with PRRSV-EGFP at an MOI of 0.1 for the indicated times and then fixed for immunofluorescent staining of NF-κB p65 (red). Simultaneously, PRRSV-EGFP (green) was also detected by immunofluorescence microscopy. Nucleus (blue) was stained with DAPI. Bar, 25 μm. (C and D) Marc-145 cells or PAMs were mock infected or infected with PRRSV (MOI of 0.1) for the indicated times, and cells were lysed for Western blotting to examine NF-κB p-IκBα and PRRSV N protein. (E and F) The endogenous expression of heparanase both at the mRNA level and at the protein level was determined in cells treated with or without LPS (NF-κB inducer) for 12 h. NF-κB p-IκBα was also determined by Western blotting to show the effect of LPS. (G and H) Cells were mock inoculated or inoculated with PRRSV (MOI of 0.1) in the presence or absence of BAY11-7082 (NF-κB inhibitor, 5 μM) for 24 h, and then heparanase expression was analyzed by qRT-PCR and Western blotting. Simultaneously, cells were also lysed for NF-κB p-IκBα detection to show the effect of BAY11-7082. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Journal: Journal of Virology

    Article Title: Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

    doi: 10.1128/JVI.00625-17

    Figure Lengend Snippet: Mechanism of heparanase upregulation after PRRSV infection. (A) Marc-145 cells were inoculated with PRRSV (MOI of 0.1) and then harvested for qRT-PCR analysis of NF-κB p65 expression at 6, 12, and 24 hpi. (B) Cells were mock infected or infected with PRRSV-EGFP at an MOI of 0.1 for the indicated times and then fixed for immunofluorescent staining of NF-κB p65 (red). Simultaneously, PRRSV-EGFP (green) was also detected by immunofluorescence microscopy. Nucleus (blue) was stained with DAPI. Bar, 25 μm. (C and D) Marc-145 cells or PAMs were mock infected or infected with PRRSV (MOI of 0.1) for the indicated times, and cells were lysed for Western blotting to examine NF-κB p-IκBα and PRRSV N protein. (E and F) The endogenous expression of heparanase both at the mRNA level and at the protein level was determined in cells treated with or without LPS (NF-κB inducer) for 12 h. NF-κB p-IκBα was also determined by Western blotting to show the effect of LPS. (G and H) Cells were mock inoculated or inoculated with PRRSV (MOI of 0.1) in the presence or absence of BAY11-7082 (NF-κB inhibitor, 5 μM) for 24 h, and then heparanase expression was analyzed by qRT-PCR and Western blotting. Simultaneously, cells were also lysed for NF-κB p-IκBα detection to show the effect of BAY11-7082. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Article Snippet: For Western blot experiments, the primary antibodies anti-PRRSV N protein MAb (1:2,000; Jeno Biotech, Inc., Republic of Korea), anti-heparanase MAb (1:1,000; Abcam), anti-NF-κB p-IκBα MAb (1:2,000; Cell Signal Technology), anti-cathepsin L (1:1,000; Abcam), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) MAb (1:2,000; Cell Signal Technology), horseradish peroxidase-conjugated anti-mouse IgG antibody, and anti-rabbit IgG antibody (1:2,000; Cell Signal Technology) were used.

    Techniques: Infection, Quantitative RT-PCR, Expressing, Staining, Immunofluorescence, Microscopy, Western Blot

    Heparanase overexpression enhances PRRSV replication and release. (A and B) Marc-145 cells were transfected with the heparanase expression plasmid pcDNA3.1-heparanase or the pcDNA3.1 control, and cells were harvested 36 h later to determine the transcript (A) and protein (B) levels of heparanase by qRT-PCR and Western blot analysis. (C to I) Cells were transfected with the heparanase expression plasmid pcDNA3.1-heparanase or the pcDNA3.1 control for 36 h and then infected with PRRSV (MOI of 0.1). At 24, 36, and 48 hpi, cell surface expression levels of HS (C) and PRRSV N protein (G to I) were determined by flow cytometry and Western blotting, respectively. Mean fluorescence intensity measurements ( x axis, log 10 fluorescence) were based on flow cytometry results (D to F). (J and K) Simultaneously, the supernatants were harvested to measure viral titers (J) and extracellular viral particles (K) using TCID 50 and qRT-PCR analysis, respectively, at the indicated time points. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Journal: Journal of Virology

    Article Title: Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

    doi: 10.1128/JVI.00625-17

    Figure Lengend Snippet: Heparanase overexpression enhances PRRSV replication and release. (A and B) Marc-145 cells were transfected with the heparanase expression plasmid pcDNA3.1-heparanase or the pcDNA3.1 control, and cells were harvested 36 h later to determine the transcript (A) and protein (B) levels of heparanase by qRT-PCR and Western blot analysis. (C to I) Cells were transfected with the heparanase expression plasmid pcDNA3.1-heparanase or the pcDNA3.1 control for 36 h and then infected with PRRSV (MOI of 0.1). At 24, 36, and 48 hpi, cell surface expression levels of HS (C) and PRRSV N protein (G to I) were determined by flow cytometry and Western blotting, respectively. Mean fluorescence intensity measurements ( x axis, log 10 fluorescence) were based on flow cytometry results (D to F). (J and K) Simultaneously, the supernatants were harvested to measure viral titers (J) and extracellular viral particles (K) using TCID 50 and qRT-PCR analysis, respectively, at the indicated time points. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Article Snippet: For Western blot experiments, the primary antibodies anti-PRRSV N protein MAb (1:2,000; Jeno Biotech, Inc., Republic of Korea), anti-heparanase MAb (1:1,000; Abcam), anti-NF-κB p-IκBα MAb (1:2,000; Cell Signal Technology), anti-cathepsin L (1:1,000; Abcam), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) MAb (1:2,000; Cell Signal Technology), horseradish peroxidase-conjugated anti-mouse IgG antibody, and anti-rabbit IgG antibody (1:2,000; Cell Signal Technology) were used.

    Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Infection, Flow Cytometry, Cytometry, Fluorescence

    Cathepsin L upregulation after PRRSV infection contributes to the activation of heparanase. (A and B) Marc-145 cells were mock infected or infected with PRRSV-EGFP at an MOI of 0.1 for the indicated times or at indicated MOIs for 4 h; cathepsin L proteolytic activity (red) in cells was assessed by a Magic Red cathepsin L detection kit, and PRRSV-EGFP (green) cells were then examined by immunofluorescence microscopy. Nucleus (blue) was stained with DAPI. Bar, 300 μm. (C to F) PRRSV-infected cells were also collected to analyze cathepsin L expression using qRT-PCR (C and D), and cathepsin L and PRRSV N protein were detected by Western blot analysis (E and F). (G to K) Cells were infected with PRRSV at an MOI of 0.1 in the presence or absence of cathepsin L inhibitor (10 μM) for 24 or 36 h. The cells were harvested for heparanase and cathepsin L analysis using antibodies against heparanase and cathepsin L by Western blotting (G and H). Additionally, cells were stained for cellular surface expression of HS with anti-human HS MAb 10E4 and then determined by flow cytometry (I). Mean fluorescence intensity measurements ( x axis, log 10 fluorescence) were based on flow cytometry results (J and K). All values are representative of three independent experiments. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Journal: Journal of Virology

    Article Title: Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

    doi: 10.1128/JVI.00625-17

    Figure Lengend Snippet: Cathepsin L upregulation after PRRSV infection contributes to the activation of heparanase. (A and B) Marc-145 cells were mock infected or infected with PRRSV-EGFP at an MOI of 0.1 for the indicated times or at indicated MOIs for 4 h; cathepsin L proteolytic activity (red) in cells was assessed by a Magic Red cathepsin L detection kit, and PRRSV-EGFP (green) cells were then examined by immunofluorescence microscopy. Nucleus (blue) was stained with DAPI. Bar, 300 μm. (C to F) PRRSV-infected cells were also collected to analyze cathepsin L expression using qRT-PCR (C and D), and cathepsin L and PRRSV N protein were detected by Western blot analysis (E and F). (G to K) Cells were infected with PRRSV at an MOI of 0.1 in the presence or absence of cathepsin L inhibitor (10 μM) for 24 or 36 h. The cells were harvested for heparanase and cathepsin L analysis using antibodies against heparanase and cathepsin L by Western blotting (G and H). Additionally, cells were stained for cellular surface expression of HS with anti-human HS MAb 10E4 and then determined by flow cytometry (I). Mean fluorescence intensity measurements ( x axis, log 10 fluorescence) were based on flow cytometry results (J and K). All values are representative of three independent experiments. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Article Snippet: For Western blot experiments, the primary antibodies anti-PRRSV N protein MAb (1:2,000; Jeno Biotech, Inc., Republic of Korea), anti-heparanase MAb (1:1,000; Abcam), anti-NF-κB p-IκBα MAb (1:2,000; Cell Signal Technology), anti-cathepsin L (1:1,000; Abcam), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) MAb (1:2,000; Cell Signal Technology), horseradish peroxidase-conjugated anti-mouse IgG antibody, and anti-rabbit IgG antibody (1:2,000; Cell Signal Technology) were used.

    Techniques: Infection, Activation Assay, Activity Assay, Immunofluorescence, Microscopy, Staining, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Cytometry, Fluorescence

    Heparanase knockdown inhibits PRRSV replication and release. (A and B) Marc-145 cells were transfected with heparanase-specific siRNAs at a final concentration of 50 nM for 36 h and then lysed for qRT-PCR to examine the heparanase mRNA level (A) or for Western blotting to examine heparanase protein levels (B). (C to F) Cells were transfected with heparanase-specific siRNAs for 36 h, followed by infection with PRRSV at an MOI of 0.1. At 24 hpi, cells were collected for flow cytometry analysis of HS cell surface expression using anti-human HS MAb 10E4 (C) or for PRRSV N protein detection by Western blot analysis (E). Mean fluorescence intensity measurements were based on flow cytometry results (D). The level of viral N protein was quantified by measuring band intensities and normalized with respect to the amount of GAPDH (F). (G and H) Simultaneously, the supernatants were harvested to measure viral titers (G) and extracellular viral particles (H) using TCID 50 and qRT-PCR analysis, respectively. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Journal: Journal of Virology

    Article Title: Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

    doi: 10.1128/JVI.00625-17

    Figure Lengend Snippet: Heparanase knockdown inhibits PRRSV replication and release. (A and B) Marc-145 cells were transfected with heparanase-specific siRNAs at a final concentration of 50 nM for 36 h and then lysed for qRT-PCR to examine the heparanase mRNA level (A) or for Western blotting to examine heparanase protein levels (B). (C to F) Cells were transfected with heparanase-specific siRNAs for 36 h, followed by infection with PRRSV at an MOI of 0.1. At 24 hpi, cells were collected for flow cytometry analysis of HS cell surface expression using anti-human HS MAb 10E4 (C) or for PRRSV N protein detection by Western blot analysis (E). Mean fluorescence intensity measurements were based on flow cytometry results (D). The level of viral N protein was quantified by measuring band intensities and normalized with respect to the amount of GAPDH (F). (G and H) Simultaneously, the supernatants were harvested to measure viral titers (G) and extracellular viral particles (H) using TCID 50 and qRT-PCR analysis, respectively. Data are representative of the results of three independent experiments (means ± SE). Significant differences from results with the control group are indicated as follows: *, P

    Article Snippet: For Western blot experiments, the primary antibodies anti-PRRSV N protein MAb (1:2,000; Jeno Biotech, Inc., Republic of Korea), anti-heparanase MAb (1:1,000; Abcam), anti-NF-κB p-IκBα MAb (1:2,000; Cell Signal Technology), anti-cathepsin L (1:1,000; Abcam), anti-glyceraldehyde phosphate dehydrogenase (GAPDH) MAb (1:2,000; Cell Signal Technology), horseradish peroxidase-conjugated anti-mouse IgG antibody, and anti-rabbit IgG antibody (1:2,000; Cell Signal Technology) were used.

    Techniques: Transfection, Concentration Assay, Quantitative RT-PCR, Western Blot, Infection, Flow Cytometry, Cytometry, Expressing, Fluorescence

    Abrogation of the perlecan binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.

    Journal: The Journal of Biological Chemistry

    Article Title: Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity *

    doi: 10.1074/jbc.M110.177865

    Figure Lengend Snippet: Abrogation of the perlecan binding activity of activin A by deletion of the inhibin β A subunit-specific region harboring Clusters 2 and 3. A , amino acid sequence alignment between the inhibin β A - and β B -subunits. The peptide segment Lys 259 -Glu 284 containing Clusters 2 and 3 in the inhibin β A -subunit is absent from the inhibin β B -subunit (highlighted in italics ). The conserved amino acid residues are shaded in gray. B , alignment of basic amino acid clusters in the amino acid sequences of the inhibin β A -subunits from different species. The amino acid sequence of the peptide segment Lys 259 -Glu 284 harboring Clusters 2 and 3 is well conserved among the different species. The clusters of basic amino acid residues are shown in white letters on black whereas acidic amino acid residues are shaded in gray. C , the perlecan binding activities of recombinant activin A with a His 6 tag at either the N terminus ( N-His tag ) or C terminus ( C-His tag ) and those with deletion of the Lys 259 -Gly 277 segment (designated ΔK 259 -G 277 ) were determined by solid-phase binding assays. The SDS-PAGE profiles of purified activin A with and without deletion of Clusters 2 and 3 are shown in the inset . Note that the pro-regions of intact and mutant activin A differ in their sizes because of the presence or absence of the Lys 259 -Gly 277 segment.

    Article Snippet: Perlecan was purified from conditioned medium of JAR human choriocarcinoma cells by immunoaffinity chromatography using an anti-perlecan mAb (kindly provided by Dr. Masahiko Katayama, Eisai Co. Ltd., Tsukuba, Japan).

    Techniques: Binding Assay, Activity Assay, Sequencing, Recombinant, SDS Page, Purification, Mutagenesis