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heparan sulfate degradation  (AMS Biotechnology)


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    AMS Biotechnology heparan sulfate degradation
    Heparan Sulfate Degradation, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ams%2Egag-hs01/Heparan+Sulfate/pmc08192744-177-17-9
    Average 96 stars, based on 102 article reviews
    heparan sulfate degradation - by Bioz Stars, 2026-10
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    Related Articles

    Staining:

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection
    Article Snippet: The cells were analyzed using a FACS Canto II (BD Biosciences) or a DxFLEX (Beckman Coulter) flow cytometer, and data were processed using WinMDI or Kaluza (Beckman Coulter) software. .. To detect HS, the cells were stained with biotinylated mouse anti-heparan sulfate antibody (10E4 epitope) (370255-B, amsbio) or biotinylated mouse IgMκ isotype control (401621, BioLegend), followed by PE-conjugated streptavidin (405203, BioLegend) staining. .. For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Article Title: Enhanced lysosomal exocytosis and altered growth factor signaling are associated with cartilage pathology in a model of mucopolysaccharidosis type IVA
    Article Snippet: 20 μm sections were cut on a Leica cryostat. .. Primary antibodies used for staining included an anti-GFP antibody (1:100; 31 A-21311, Thermo Fisher Scientific), an anti-heparan sulfate (1:100; clone F58-10E4, Amsbio) and an anti-chondroitin sulfate antibody (1:100; MA1-83055, Invitrogen, Thermo Fisher Scientific). .. Secondary antibodies utilized included anti-mouse Alexa 568 (1:400; 31A-11031, Invitrogen) and anti-GFPAlexa488 (1:200; 31FAB42401G, R&D Systems).

    Control:

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection
    Article Snippet: The cells were analyzed using a FACS Canto II (BD Biosciences) or a DxFLEX (Beckman Coulter) flow cytometer, and data were processed using WinMDI or Kaluza (Beckman Coulter) software. .. To detect HS, the cells were stained with biotinylated mouse anti-heparan sulfate antibody (10E4 epitope) (370255-B, amsbio) or biotinylated mouse IgMκ isotype control (401621, BioLegend), followed by PE-conjugated streptavidin (405203, BioLegend) staining. .. For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Incubation:

    Article Title: Endoplasmic reticulum stress induced autophagy alters cellular processing of cationic lipid delivered siRNAs.
    Article Snippet: Complexes were made to achieve a final concentration of 1.75 μg/mL LF2K (Invitrogen, 11,668,019) and 150 nM siRNA (Horizon Discovery, Guide strand sequence: GGC UAC GUC CAG GAG CGC AUU) in 50 μL of Opti-MEM (Gibco, 31,985,062) and 500 μL of DMEM. .. Cells were incubated with the transfection solution for 4 h and then washed with 50 μg/mL heparan sulfate (AMSBIO, GAG-HS01) in 1 mL DPBS (Gibco, 14,190,144) for 10 min. ..

    Article Title: Early Transcriptomic Response of Human Iris Stromal Cells During Herpes Simplex Virus Entry Reveals Interplay Between Cell Glycocalyx and Viral Exploitation
    Article Snippet: Samples were then blocked using 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) in PBS with 0.2% Tween-20 (PBS-T). .. After blocking, coverslips were washed again and incubated with mouse anti-Heparan Sulfate (1:500 dilution, Amsbio, Cambridge, MA F58-10E4, catalog no: 370255-S), anti-CD138 (syndecan-1) monoclonal antibody (1:200 dilution, ThermoFisher, Waltham, MA, DL-101, catalog no: 14-1389-82), anti-syndecan-2 polyclonal antibody (1:125 dilution, ThermoFisher, Waltham, MA, USA, catalog no: 36-6200). .. Coverslips were then washed with PBS-T three times and incubated with the appropriate Alexa Fluor 594 secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h in 1% BSA in PBS-T.

    Article Title: Two-photon microscopy of brain endothelial glycocalyx uncovers spatial heterogeneity, vesicular transport, and lectin-binding kinetics in the living brain
    Article Snippet: .. Brain sections were incubated with primary antibody diluted in 2.5% BSA in PBS (anti-heparan sulfate, 1:100, Amsbio EU, clone 10E4) overnight at 4°C, washed in 1× PBS, and incubated with secondary antibody, Alexa Fluor 488-conjugated goat anti-mouse (1:500, Molecular Probes, Thermo Scientific, EU). .. After washing, the sections were mounted using ProLong Gold Antifade Reagent with DAPI (Molecular Probes, Thermo Scientific, EU).

    Article Title: ADAM10 tailors extracellular vesicles for content transfer rather than signaling by contact
    Article Snippet: Antibodies directed against SDC1 intracellular domain (D4Y7H) was from (cell signaling #12922, dilution 1/1000), GFP (A11122) from Thermofisher (dilution 1/1000), ADAM17 (abcam, #ab39162, 1/1000 or cell signaling #3976, 1/1000), ADAM10 (abcam, #ab1997, 1/1 000), GAPDH (Proteintech # 10494-1-AP), Flotillin-1 (BD Biosciences, #X11669), Ephrin B3 (santa cruz, # sc-514139), PTK7 (Proteintech, #17799), E-cadherin (R&D Systems, #AF748), BCAM (R&D Systems, #BAF148), STAT3 124H6 (cell signaling #9139), p-STAT3 (cell signaling #9131). .. To enzymatically digest the glycosaminoglycans (GAGs) on SDC, including heparan sulfate (HS) and chondroitin sulfate (CS), nine volumes of cell extract or EVs were incubated with one volume of 10x heparitinase reaction buffer (1M NaCl, 500mM hepes pH 7,0, 10mM CaCl2, 1% TX100, Pepstatin and Leupeptin, both at a concentration of 10 μg/mL), heparitinase (0.4 milliIU, amsbio), and chondroitinase (20milliunits, amsbio). ..

    Article Title: Early Transcriptomic Response of Human Iris Stromal Cells During Herpes Simplex Virus Entry Reveals Interplay Between Cell Glycocalyx and Viral Exploitation
    Article Snippet: Samples were then blocked using 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) in PBS with 0.2% Tween-20 (PBS-T). .. After blocking, coverslips were washed again and incubated with mouse anti-Heparan Sulfate (1:500 dilution, Amsbio, Cambridge, MA F58-10E4, catalog no: 370255-S), anti-CD138 (syndecan-1) monoclonal antibody (1:200 dilution, ThermoFisher, Waltham, MA, DL-101, catalog no: 14-1389-82), anti-syndecan-2 polyclonal antibody (1:125 dilution, ThermoFisher, Waltham, MA, USA, catalog no: 36-6200). .. Coverslips were then washed with PBS-T three times and incubated with the appropriate Alexa Fluor 594 secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h in 1% BSA in PBS-T.

    Transfection:

    Article Title: Endoplasmic reticulum stress induced autophagy alters cellular processing of cationic lipid delivered siRNAs.
    Article Snippet: Complexes were made to achieve a final concentration of 1.75 μg/mL LF2K (Invitrogen, 11,668,019) and 150 nM siRNA (Horizon Discovery, Guide strand sequence: GGC UAC GUC CAG GAG CGC AUU) in 50 μL of Opti-MEM (Gibco, 31,985,062) and 500 μL of DMEM. .. Cells were incubated with the transfection solution for 4 h and then washed with 50 μg/mL heparan sulfate (AMSBIO, GAG-HS01) in 1 mL DPBS (Gibco, 14,190,144) for 10 min. ..

    Blocking Assay:

    Article Title: Early Transcriptomic Response of Human Iris Stromal Cells During Herpes Simplex Virus Entry Reveals Interplay Between Cell Glycocalyx and Viral Exploitation
    Article Snippet: Samples were then blocked using 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) in PBS with 0.2% Tween-20 (PBS-T). .. After blocking, coverslips were washed again and incubated with mouse anti-Heparan Sulfate (1:500 dilution, Amsbio, Cambridge, MA F58-10E4, catalog no: 370255-S), anti-CD138 (syndecan-1) monoclonal antibody (1:200 dilution, ThermoFisher, Waltham, MA, DL-101, catalog no: 14-1389-82), anti-syndecan-2 polyclonal antibody (1:125 dilution, ThermoFisher, Waltham, MA, USA, catalog no: 36-6200). .. Coverslips were then washed with PBS-T three times and incubated with the appropriate Alexa Fluor 594 secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h in 1% BSA in PBS-T.

    Article Title: Early Transcriptomic Response of Human Iris Stromal Cells During Herpes Simplex Virus Entry Reveals Interplay Between Cell Glycocalyx and Viral Exploitation
    Article Snippet: Samples were then blocked using 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) in PBS with 0.2% Tween-20 (PBS-T). .. After blocking, coverslips were washed again and incubated with mouse anti-Heparan Sulfate (1:500 dilution, Amsbio, Cambridge, MA F58-10E4, catalog no: 370255-S), anti-CD138 (syndecan-1) monoclonal antibody (1:200 dilution, ThermoFisher, Waltham, MA, DL-101, catalog no: 14-1389-82), anti-syndecan-2 polyclonal antibody (1:125 dilution, ThermoFisher, Waltham, MA, USA, catalog no: 36-6200). .. Coverslips were then washed with PBS-T three times and incubated with the appropriate Alexa Fluor 594 secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h in 1% BSA in PBS-T.

    Concentration Assay:

    Article Title: ADAM10 tailors extracellular vesicles for content transfer rather than signaling by contact
    Article Snippet: Antibodies directed against SDC1 intracellular domain (D4Y7H) was from (cell signaling #12922, dilution 1/1000), GFP (A11122) from Thermofisher (dilution 1/1000), ADAM17 (abcam, #ab39162, 1/1000 or cell signaling #3976, 1/1000), ADAM10 (abcam, #ab1997, 1/1 000), GAPDH (Proteintech # 10494-1-AP), Flotillin-1 (BD Biosciences, #X11669), Ephrin B3 (santa cruz, # sc-514139), PTK7 (Proteintech, #17799), E-cadherin (R&D Systems, #AF748), BCAM (R&D Systems, #BAF148), STAT3 124H6 (cell signaling #9139), p-STAT3 (cell signaling #9131). .. To enzymatically digest the glycosaminoglycans (GAGs) on SDC, including heparan sulfate (HS) and chondroitin sulfate (CS), nine volumes of cell extract or EVs were incubated with one volume of 10x heparitinase reaction buffer (1M NaCl, 500mM hepes pH 7,0, 10mM CaCl2, 1% TX100, Pepstatin and Leupeptin, both at a concentration of 10 μg/mL), heparitinase (0.4 milliIU, amsbio), and chondroitinase (20milliunits, amsbio). ..

    other:

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection.
    Article Snippet: Cells were detached using Accutase (12679-54, Nacalai Tesque) and incubated with primary antibodies for 30 minutes on ice, followed by incubation with secondary antibodies for an additional 30 minutes on ice if necessary.



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    A. Volcano plot illustrating the proteins associated with GFP-SDC4 according to differential proteomics, with log2 fold change (x-axis) and -log10 (p-value) (y-axis). Data were obtained by GFP-pulldown from total extracts of MCF7 cells overexpressing GFP-SDC4 compared to MCF7 cells overexpressing GFP alone. Data result from three different experiments processed three times. SDC4 (the bait) is represented in green, ADAM10 and ADAM17, the two metalloproteases associated with SDC4 are represented in red. The other proteins are represented in grey. The proteins showing non-significant differences are represented below the dotted lines (threshold at 1 for the x-axis, difference =2 and 1.5 for y-axis; p-value = 0.3). B. Co-immunoprecipitation experiments from MCF7 cell lysates, confirming the association of endogenous ADAM10 and ADAM17 with GFP-SDC4, but not GFP. The GFP precipitates (IP) were subjected to Western blot with antibodies recognizing proteins as indicated on the right. C . Left: Scheme illustrating the complexity of SDC4 structure and processing. (Upper part) SDC4 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the <t>heparan</t> <t>sulfate</t> type (green). The trimming of GAG chains by specific enzymes is necessary for the FL protein to migrate at a discrete band in SDS-PAGE and to be easily detectable by immunoblotting. (Lower part) SDC4 core protein can be cleaved by proteases generating two main fragments: an N-terminal fragment comprising most of the GAG-substituted extracellular domain (ECD) and a C-terminal fragment (CTF) comprising the remainder of the ECD, the membrane-spanning and the cytoplasmic domain. Right: Western blot illustrating the signals obtained, after GAG-digestion, for FL SDC4 and SDC4 CTF in the cells and the sEV enriched fraction obtained after differential ultracentrifugation of the conditioned extracellular media. Signals were obtained with an antibody recognizing the intracellular domain of SDC4. Note that the FL form of SDC4 (SDC4-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC4-CTF is abundant and the SDC4-FL is barely detectable in sEVs. D. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC4 FL and CTF abundance in cells and sEVs by Western blot after GAG-digestion. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. Statistical analysis was performed using the Kruskal-Wallis one-way non-parametric ANOVA test (* P < 0.05, *** P < 0.001, n.s. non-significant). E . sEVs secreted by MCF7 cells inhibited for ADAM10 activity, (GI254023X) versus controls (DMSO) were isolated from conditioned media by differential ultracentrifugation. sEVs were analyzed by Western blot after GAG-digestion to evaluate the levels of SDC4 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. ** P < 0.01 (unpaired non-parametric Mann–Whitney test).
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    A. Volcano plot illustrating the proteins associated with GFP-SDC4 according to differential proteomics, with log2 fold change (x-axis) and -log10 (p-value) (y-axis). Data were obtained by GFP-pulldown from total extracts of MCF7 cells overexpressing GFP-SDC4 compared to MCF7 cells overexpressing GFP alone. Data result from three different experiments processed three times. SDC4 (the bait) is represented in green, ADAM10 and ADAM17, the two metalloproteases associated with SDC4 are represented in red. The other proteins are represented in grey. The proteins showing non-significant differences are represented below the dotted lines (threshold at 1 for the x-axis, difference =2 and 1.5 for y-axis; p-value = 0.3). B. Co-immunoprecipitation experiments from MCF7 cell lysates, confirming the association of endogenous ADAM10 and ADAM17 with GFP-SDC4, but not GFP. The GFP precipitates (IP) were subjected to Western blot with antibodies recognizing proteins as indicated on the right. C . Left: Scheme illustrating the complexity of SDC4 structure and processing. (Upper part) SDC4 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the <t>heparan</t> <t>sulfate</t> type (green). The trimming of GAG chains by specific enzymes is necessary for the FL protein to migrate at a discrete band in SDS-PAGE and to be easily detectable by immunoblotting. (Lower part) SDC4 core protein can be cleaved by proteases generating two main fragments: an N-terminal fragment comprising most of the GAG-substituted extracellular domain (ECD) and a C-terminal fragment (CTF) comprising the remainder of the ECD, the membrane-spanning and the cytoplasmic domain. Right: Western blot illustrating the signals obtained, after GAG-digestion, for FL SDC4 and SDC4 CTF in the cells and the sEV enriched fraction obtained after differential ultracentrifugation of the conditioned extracellular media. Signals were obtained with an antibody recognizing the intracellular domain of SDC4. Note that the FL form of SDC4 (SDC4-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC4-CTF is abundant and the SDC4-FL is barely detectable in sEVs. D. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC4 FL and CTF abundance in cells and sEVs by Western blot after GAG-digestion. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. Statistical analysis was performed using the Kruskal-Wallis one-way non-parametric ANOVA test (* P < 0.05, *** P < 0.001, n.s. non-significant). E . sEVs secreted by MCF7 cells inhibited for ADAM10 activity, (GI254023X) versus controls (DMSO) were isolated from conditioned media by differential ultracentrifugation. sEVs were analyzed by Western blot after GAG-digestion to evaluate the levels of SDC4 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. ** P < 0.01 (unpaired non-parametric Mann–Whitney test).
    Resource Source Identifier Antibodies Mouse Monoclonal Anti Heparan Sulfate 10e4 Amsbio Rrid Ab 10013601 Goat Anti Mouse Igg, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A. Volcano plot illustrating the proteins associated with GFP-SDC4 according to differential proteomics, with log2 fold change (x-axis) and -log10 (p-value) (y-axis). Data were obtained by GFP-pulldown from total extracts of MCF7 cells overexpressing GFP-SDC4 compared to MCF7 cells overexpressing GFP alone. Data result from three different experiments processed three times. SDC4 (the bait) is represented in green, ADAM10 and ADAM17, the two metalloproteases associated with SDC4 are represented in red. The other proteins are represented in grey. The proteins showing non-significant differences are represented below the dotted lines (threshold at 1 for the x-axis, difference =2 and 1.5 for y-axis; p-value = 0.3). B. Co-immunoprecipitation experiments from MCF7 cell lysates, confirming the association of endogenous ADAM10 and ADAM17 with GFP-SDC4, but not GFP. The GFP precipitates (IP) were subjected to Western blot with antibodies recognizing proteins as indicated on the right. C . Left: Scheme illustrating the complexity of SDC4 structure and processing. (Upper part) SDC4 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the heparan sulfate type (green). The trimming of GAG chains by specific enzymes is necessary for the FL protein to migrate at a discrete band in SDS-PAGE and to be easily detectable by immunoblotting. (Lower part) SDC4 core protein can be cleaved by proteases generating two main fragments: an N-terminal fragment comprising most of the GAG-substituted extracellular domain (ECD) and a C-terminal fragment (CTF) comprising the remainder of the ECD, the membrane-spanning and the cytoplasmic domain. Right: Western blot illustrating the signals obtained, after GAG-digestion, for FL SDC4 and SDC4 CTF in the cells and the sEV enriched fraction obtained after differential ultracentrifugation of the conditioned extracellular media. Signals were obtained with an antibody recognizing the intracellular domain of SDC4. Note that the FL form of SDC4 (SDC4-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC4-CTF is abundant and the SDC4-FL is barely detectable in sEVs. D. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC4 FL and CTF abundance in cells and sEVs by Western blot after GAG-digestion. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. Statistical analysis was performed using the Kruskal-Wallis one-way non-parametric ANOVA test (* P < 0.05, *** P < 0.001, n.s. non-significant). E . sEVs secreted by MCF7 cells inhibited for ADAM10 activity, (GI254023X) versus controls (DMSO) were isolated from conditioned media by differential ultracentrifugation. sEVs were analyzed by Western blot after GAG-digestion to evaluate the levels of SDC4 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. ** P < 0.01 (unpaired non-parametric Mann–Whitney test).

    Journal: bioRxiv

    Article Title: ADAM10 tailors extracellular vesicles for content transfer rather than signaling by contact

    doi: 10.64898/2026.02.12.705562

    Figure Lengend Snippet: A. Volcano plot illustrating the proteins associated with GFP-SDC4 according to differential proteomics, with log2 fold change (x-axis) and -log10 (p-value) (y-axis). Data were obtained by GFP-pulldown from total extracts of MCF7 cells overexpressing GFP-SDC4 compared to MCF7 cells overexpressing GFP alone. Data result from three different experiments processed three times. SDC4 (the bait) is represented in green, ADAM10 and ADAM17, the two metalloproteases associated with SDC4 are represented in red. The other proteins are represented in grey. The proteins showing non-significant differences are represented below the dotted lines (threshold at 1 for the x-axis, difference =2 and 1.5 for y-axis; p-value = 0.3). B. Co-immunoprecipitation experiments from MCF7 cell lysates, confirming the association of endogenous ADAM10 and ADAM17 with GFP-SDC4, but not GFP. The GFP precipitates (IP) were subjected to Western blot with antibodies recognizing proteins as indicated on the right. C . Left: Scheme illustrating the complexity of SDC4 structure and processing. (Upper part) SDC4 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the heparan sulfate type (green). The trimming of GAG chains by specific enzymes is necessary for the FL protein to migrate at a discrete band in SDS-PAGE and to be easily detectable by immunoblotting. (Lower part) SDC4 core protein can be cleaved by proteases generating two main fragments: an N-terminal fragment comprising most of the GAG-substituted extracellular domain (ECD) and a C-terminal fragment (CTF) comprising the remainder of the ECD, the membrane-spanning and the cytoplasmic domain. Right: Western blot illustrating the signals obtained, after GAG-digestion, for FL SDC4 and SDC4 CTF in the cells and the sEV enriched fraction obtained after differential ultracentrifugation of the conditioned extracellular media. Signals were obtained with an antibody recognizing the intracellular domain of SDC4. Note that the FL form of SDC4 (SDC4-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC4-CTF is abundant and the SDC4-FL is barely detectable in sEVs. D. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC4 FL and CTF abundance in cells and sEVs by Western blot after GAG-digestion. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. Statistical analysis was performed using the Kruskal-Wallis one-way non-parametric ANOVA test (* P < 0.05, *** P < 0.001, n.s. non-significant). E . sEVs secreted by MCF7 cells inhibited for ADAM10 activity, (GI254023X) versus controls (DMSO) were isolated from conditioned media by differential ultracentrifugation. sEVs were analyzed by Western blot after GAG-digestion to evaluate the levels of SDC4 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. ** P < 0.01 (unpaired non-parametric Mann–Whitney test).

    Article Snippet: To enzymatically digest the glycosaminoglycans (GAGs) on SDC, including heparan sulfate (HS) and chondroitin sulfate (CS), nine volumes of cell extract or EVs were incubated with one volume of 10x heparitinase reaction buffer (1M NaCl, 500mM hepes pH 7,0, 10mM CaCl2, 1% TX100, Pepstatin and Leupeptin, both at a concentration of 10 μg/mL), heparitinase (0.4 milliIU, amsbio), and chondroitinase (20milliunits, amsbio).

    Techniques: Immunoprecipitation, Western Blot, SDS Page, Membrane, Control, Activity Assay, Isolation, MANN-WHITNEY

    A . Left: Scheme illustrating the complexity of SDC1 structure and processing. (Upper part) SDC1 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the heparan (green) and chondroitin (orange) sulfate type. The trimming of GAG chains by specific enzymes is necessary for the FL protein to migrate at a discrete band in SDS-PAGE and to be detectable by immunoblotting. (Lower part) SDC1 core protein can be cleaved by proteases generating two main fragments: an N-terminal fragment comprising most of the GAG-substituted extracellular domain (ECD) and a C-terminal fragment (CTF) comprising the remainder of the ECD, the membrane-spanning and the cytoplasmic domain. Right: Western blot illustrating the signals obtained, after GAG-digestion, for FL SDC1 and SDC1 CTF in the cells and the sEV enriched fraction obtained after differential ultracentrifugation of the conditioned extracellular media. Signals were obtained with an antibody recognizing the intracellular domain of SDC1. Note that the FL form of SDC1 (SDC1-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC1-CTF is abundant and the SDC1-FL is barely detectable in sEVs. B. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC1 FL and CTF abundance in cells and sEVs by Western blot, after GAG-digestion. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. Statistical analysis was performed using the Kruskal-Wallis one-way non-parametric ANOVA test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. non-significant). C. MCF7 cells downregulated for ADAM10 (siADAM10) and control cells (siCTRL) were treated with heparitinase and chondroitinase (GAG digestion +) or not (GAG digestion -) to evaluate SDC substitution with GAG chains. SDCs FL and CTF in cells and sEVs were analyzed by Western blot as indicated. Single blots examining the relative abundance of FL versus CTF forms of SDC1 ( D ) or SDC4 ( E ) forms are provided. F . sEVs secreted by MCF7 cells inhibited for ADAM10 activity, (GI254023X) versus controls (DMSO) were isolated from conditioned media by differential ultracentrifugation. sEVs were analyzed by Western blot after GAG-digestion to evaluate the levels of SDC1 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. non-significant (unpaired non-parametric Mann–Whitney test). n indicates the number of independent experiments; each point represents one independent experiment.

    Journal: bioRxiv

    Article Title: ADAM10 tailors extracellular vesicles for content transfer rather than signaling by contact

    doi: 10.64898/2026.02.12.705562

    Figure Lengend Snippet: A . Left: Scheme illustrating the complexity of SDC1 structure and processing. (Upper part) SDC1 full-length (FL) core protein (black) is substituted with glycosaminoglycan chains (GAG) of the heparan (green) and chondroitin (orange) sulfate type. The trimming of GAG chains by specific enzymes is necessary for the FL protein to migrate at a discrete band in SDS-PAGE and to be detectable by immunoblotting. (Lower part) SDC1 core protein can be cleaved by proteases generating two main fragments: an N-terminal fragment comprising most of the GAG-substituted extracellular domain (ECD) and a C-terminal fragment (CTF) comprising the remainder of the ECD, the membrane-spanning and the cytoplasmic domain. Right: Western blot illustrating the signals obtained, after GAG-digestion, for FL SDC1 and SDC1 CTF in the cells and the sEV enriched fraction obtained after differential ultracentrifugation of the conditioned extracellular media. Signals were obtained with an antibody recognizing the intracellular domain of SDC1. Note that the FL form of SDC1 (SDC1-FL) abounds in cell lysates, while the CTF is less abundant. On the contrary, the SDC1-CTF is abundant and the SDC1-FL is barely detectable in sEVs. B. MCF7 cells downregulated for ADAM10 (siADAM10) or ADAM17 (siADAM17) and control cells (siCTRL) were evaluated for SDC1 FL and CTF abundance in cells and sEVs by Western blot, after GAG-digestion. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. Statistical analysis was performed using the Kruskal-Wallis one-way non-parametric ANOVA test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. non-significant). C. MCF7 cells downregulated for ADAM10 (siADAM10) and control cells (siCTRL) were treated with heparitinase and chondroitinase (GAG digestion +) or not (GAG digestion -) to evaluate SDC substitution with GAG chains. SDCs FL and CTF in cells and sEVs were analyzed by Western blot as indicated. Single blots examining the relative abundance of FL versus CTF forms of SDC1 ( D ) or SDC4 ( E ) forms are provided. F . sEVs secreted by MCF7 cells inhibited for ADAM10 activity, (GI254023X) versus controls (DMSO) were isolated from conditioned media by differential ultracentrifugation. sEVs were analyzed by Western blot after GAG-digestion to evaluate the levels of SDC1 FL and CTF. Histograms represent the mean signal intensity for indicated proteins relative to the signal in control cells, ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. non-significant (unpaired non-parametric Mann–Whitney test). n indicates the number of independent experiments; each point represents one independent experiment.

    Article Snippet: To enzymatically digest the glycosaminoglycans (GAGs) on SDC, including heparan sulfate (HS) and chondroitin sulfate (CS), nine volumes of cell extract or EVs were incubated with one volume of 10x heparitinase reaction buffer (1M NaCl, 500mM hepes pH 7,0, 10mM CaCl2, 1% TX100, Pepstatin and Leupeptin, both at a concentration of 10 μg/mL), heparitinase (0.4 milliIU, amsbio), and chondroitinase (20milliunits, amsbio).

    Techniques: SDS Page, Western Blot, Membrane, Control, Activity Assay, Isolation, MANN-WHITNEY

    A-B. Western blot of the cell lysates and sEVs from HEK293 cells treated with the ADAM10 inhibitor GI254023X or DMSO as control, in the presence or absence of serum (FCS), show that ADAM10 supports the cleavage of various receptors, as indicated by the observed increase of non-cleaved receptors in the sEV of those cells, irrespectively of serum (although the absence of serum significantly reduces sEV accumulations). These control experiments were performed to ensure the pertinence of the endosomal escape assay as developed by Hyka et al., 2025 in the context of the present study. For SDCs (A), cells were treated with heparatinase and chondroitinase (GAG digestion +) or not (GAG digestion -). Heparan sulfate proteoglycans / SDCs are detected with the mAb 3G10 recognizing GAG chain stubs left after digestion ( David et al , 1992 ). C. Left. Histogram illustrating that the uptake of HiBiT-syntenin sEVs by HEK293 LgBiT cells after 4 hours of incubation is comparable regardless of treatment with the ADAM10 inhibitor GI254023X. Right. Histogram illustrating that endosomal escape of HiBit-syntenin sEVs is abolished when sEVs originate from HEK293 treated with ADAM10 inhibitor (GI254023X). Histograms represent mean signal intensities in relative light units (RLU) after addition of the nanoluciferase substrate Furimazine. D. Illustrative western blot of size exclusion chromatography (SEC) experiments for the preparation of sEVs for functional assays. Left. Syntenin, CD9, CD81, CD63, ADAM10, SDC1 and SDC4 were used as markers to select ad-hoc fractions from the conditioned media of MCF7 cells KO for ADAM10 (sEV KO) or control cells (sEV WT) as indicated. Fractions 6-9 were pooled before incubation with HUVEC cells. Right. Syntenin, CD9, CD81, CD63, SDC1 and EGFR were used as markers to select ad-hoc fractions from the conditioned media of MDA-MB-468 WT cells as indicated. Fractions 6-8 were pooled before incubation with HUVEC cells. E. The phosphorylation of selected proteins (top nine proteins showing increased phosphorylation after ADAM10-KO EV treatment) were determined using the phospho-array (R&D systems). Signals for each phosphorylated protein are presented as a pair of duplicate spots for the same exposition of 15 minutes. Average densitometric values for the phosphorylated proteins are shown in the heatmap . F. Heatmap showing log 2-fold changes of protein phosphorylation after treatment with sEV from MDA-MB-468 ADAM10 KO cells compared to control WT cells. The log 2-fold changes of protein phosphorylation after sEV KO treatment compared to control (sEV WT) were shown in the heatmap. The level of average densitometric values (Log2) are presented as a spectrum of color where white and red colors represent the lowest and the highest values in the row according to the indicated scale. The difference (diff, shown in ochre) corresponds to the difference between KO and WT conditions of the same protein, based on their respective signal intensity difference between KO and Ctrl conditions. The experiment was performed two or three times depending on the analyzed protein.

    Journal: bioRxiv

    Article Title: ADAM10 tailors extracellular vesicles for content transfer rather than signaling by contact

    doi: 10.64898/2026.02.12.705562

    Figure Lengend Snippet: A-B. Western blot of the cell lysates and sEVs from HEK293 cells treated with the ADAM10 inhibitor GI254023X or DMSO as control, in the presence or absence of serum (FCS), show that ADAM10 supports the cleavage of various receptors, as indicated by the observed increase of non-cleaved receptors in the sEV of those cells, irrespectively of serum (although the absence of serum significantly reduces sEV accumulations). These control experiments were performed to ensure the pertinence of the endosomal escape assay as developed by Hyka et al., 2025 in the context of the present study. For SDCs (A), cells were treated with heparatinase and chondroitinase (GAG digestion +) or not (GAG digestion -). Heparan sulfate proteoglycans / SDCs are detected with the mAb 3G10 recognizing GAG chain stubs left after digestion ( David et al , 1992 ). C. Left. Histogram illustrating that the uptake of HiBiT-syntenin sEVs by HEK293 LgBiT cells after 4 hours of incubation is comparable regardless of treatment with the ADAM10 inhibitor GI254023X. Right. Histogram illustrating that endosomal escape of HiBit-syntenin sEVs is abolished when sEVs originate from HEK293 treated with ADAM10 inhibitor (GI254023X). Histograms represent mean signal intensities in relative light units (RLU) after addition of the nanoluciferase substrate Furimazine. D. Illustrative western blot of size exclusion chromatography (SEC) experiments for the preparation of sEVs for functional assays. Left. Syntenin, CD9, CD81, CD63, ADAM10, SDC1 and SDC4 were used as markers to select ad-hoc fractions from the conditioned media of MCF7 cells KO for ADAM10 (sEV KO) or control cells (sEV WT) as indicated. Fractions 6-9 were pooled before incubation with HUVEC cells. Right. Syntenin, CD9, CD81, CD63, SDC1 and EGFR were used as markers to select ad-hoc fractions from the conditioned media of MDA-MB-468 WT cells as indicated. Fractions 6-8 were pooled before incubation with HUVEC cells. E. The phosphorylation of selected proteins (top nine proteins showing increased phosphorylation after ADAM10-KO EV treatment) were determined using the phospho-array (R&D systems). Signals for each phosphorylated protein are presented as a pair of duplicate spots for the same exposition of 15 minutes. Average densitometric values for the phosphorylated proteins are shown in the heatmap . F. Heatmap showing log 2-fold changes of protein phosphorylation after treatment with sEV from MDA-MB-468 ADAM10 KO cells compared to control WT cells. The log 2-fold changes of protein phosphorylation after sEV KO treatment compared to control (sEV WT) were shown in the heatmap. The level of average densitometric values (Log2) are presented as a spectrum of color where white and red colors represent the lowest and the highest values in the row according to the indicated scale. The difference (diff, shown in ochre) corresponds to the difference between KO and WT conditions of the same protein, based on their respective signal intensity difference between KO and Ctrl conditions. The experiment was performed two or three times depending on the analyzed protein.

    Article Snippet: To enzymatically digest the glycosaminoglycans (GAGs) on SDC, including heparan sulfate (HS) and chondroitin sulfate (CS), nine volumes of cell extract or EVs were incubated with one volume of 10x heparitinase reaction buffer (1M NaCl, 500mM hepes pH 7,0, 10mM CaCl2, 1% TX100, Pepstatin and Leupeptin, both at a concentration of 10 μg/mL), heparitinase (0.4 milliIU, amsbio), and chondroitinase (20milliunits, amsbio).

    Techniques: Western Blot, Control, Incubation, Size-exclusion Chromatography, Functional Assay, Phospho-proteomics