Journal: Biomolecules

Article Title: Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

doi: 10.3390/biom5031741

Figure Lengend Snippet: Comparison of protein profiles of MBs and EVs from OVMz cells. ( A ) Immunoblotting of cellular extracts (CE), post-100,000 g supernatant from MBs isolation (S), MBs and EVs. Ten µg of total protein were applied per lane with the exception of EVs in the incubation with LGALS3BP where three µg of total protein were used. Detection was by the chemiluminescent method. Results were representative of three experiments; ( B ) SDS-PAGE analysis of proteins of MBs and EVs. Ten µg of protein were applied per lane. Protein detection was with Coomassie R-250.

Article Snippet: For each immunoprecipitation, 20 μL aliquot of Protein A/G-agarose beads (Santa Cruz Biotechnologies) were incubated with 5 μL of goat anti-human LGALS3BP polyclonal antibody (R&D) for 20 min, at 4 °C, with constant rotation, in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton-X 100, 0.02% protease inhibitors cocktail, Complete, Roche Biodiagnostics GmbH).

Techniques: Western Blot, Isolation, Incubation, SDS Page

Journal: Biomolecules

Article Title: Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

doi: 10.3390/biom5031741

Figure Lengend Snippet: List of proteins identified in EVs and MBs from OVMz cells using MALDI-TOF/TOF after SDS-PAGE separation using MALDI-TOF/TOF. Bands were excised from the gel shown in Figure 2 B.

Article Snippet: For each immunoprecipitation, 20 μL aliquot of Protein A/G-agarose beads (Santa Cruz Biotechnologies) were incubated with 5 μL of goat anti-human LGALS3BP polyclonal antibody (R&D) for 20 min, at 4 °C, with constant rotation, in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton-X 100, 0.02% protease inhibitors cocktail, Complete, Roche Biodiagnostics GmbH).

Techniques: Sequencing

Journal: Biomolecules

Article Title: Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

doi: 10.3390/biom5031741

Figure Lengend Snippet: Deglycosylation of immunoprecipitated LGALS3BP. LGALS3BP was deglycosylated with Endo H, PNGase F, and sialidase from V. cholerae after immunoprecipitation from EVs (Ctr). The input EVs contained three µg of total protein. As control for the digestion the immunoprecipitate was incubated with the corresponding buffer (bufH for Endo H, bufF for PNGase F and bufS for sialidase). The controls of the immunoprecipitation without EVs ( w / o EVs) and without antibody ( w / o Ab) were also shown in the second panel. The blots are representative of two (Endo H) or four (PNGase F and sialidase) experiments. Immunoglobulin G bands are represented with *.

Article Snippet: For each immunoprecipitation, 20 μL aliquot of Protein A/G-agarose beads (Santa Cruz Biotechnologies) were incubated with 5 μL of goat anti-human LGALS3BP polyclonal antibody (R&D) for 20 min, at 4 °C, with constant rotation, in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton-X 100, 0.02% protease inhibitors cocktail, Complete, Roche Biodiagnostics GmbH).

Techniques: Immunoprecipitation, Incubation

Journal: Journal of Extracellular Vesicles

Article Title: Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals

doi: 10.3402/jev.v4.27378

Figure Lengend Snippet: Hierarchical clustering of plasma data sets. The 13 data sets of proteomic data on plasma-derived exosomes were clustered after removal of proteins that were detected in only one of the data sets. For the remaining 182 proteins, a distance matrix based on their presence/absence was built using a binary method. The calculated distances were used to generate the hierarchical cluster. The red rectangle highlights the proteins that were more frequently detected (≥9). The red arrows indicate 2 proteins, CD5 antigen-like (CD5L) and galectin-3 binding protein (LGALS3BP).

Article Snippet: The primary antibodies were used at the following dilutions: anti-CD9 at 1:10, anti-CD81 at 1:10, anti-CD5L (Abcam, Cambrigde, UK, catalogue number ab45408) at 1:100 and anti-LGALS3BP (Acris Antibodies, San Diego, CA, USA, catalogue number AM33169PU-N) at 1:1,000.

Techniques: Derivative Assay, Binding Assay

Journal: Journal of Extracellular Vesicles

Article Title: Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals

doi: 10.3402/jev.v4.27378

Figure Lengend Snippet: CD5L and LGALS3BP have similar elution profiles to the exosome marker CD81. Plasma from “Donor 1” was submitted to size-exclusion chromatography and fractions 6–12 were analysed by NTA, flow cytometry and transmission electron microscopy (TEM). (a) NTA was performed on a NanoSight LM10 (software version 3.0). For flow cytometry, samples were coupled to 4 µm beads and incubated with primary antibodies against CD81 (1:10), CD5L (1:100) or LGALS3BP (1:1,000). The secondary antibody was conjugated to Alexa 488 was used at a 1:1,000 dilution. MFI: mean fluorescence intensity. (b) Fraction 6 from size-exclusion chromatography was submitted to cryo-EM and (c) immunostained with anti-CD5L antibodies conjugated to gold spheres of 20 nm.

Article Snippet: The primary antibodies were used at the following dilutions: anti-CD9 at 1:10, anti-CD81 at 1:10, anti-CD5L (Abcam, Cambrigde, UK, catalogue number ab45408) at 1:100 and anti-LGALS3BP (Acris Antibodies, San Diego, CA, USA, catalogue number AM33169PU-N) at 1:1,000.

Techniques: Marker, Size-exclusion Chromatography, Flow Cytometry, Transmission Assay, Electron Microscopy, Software, Incubation, Fluorescence, Cryo-EM Sample Prep

Journal: Biomolecules

Article Title: Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

doi: 10.3390/biom5031741

Figure Lengend Snippet: Comparison of protein profiles of MBs and EVs from OVMz cells. ( A ) Immunoblotting of cellular extracts (CE), post-100,000 g supernatant from MBs isolation (S), MBs and EVs. Ten µg of total protein were applied per lane with the exception of EVs in the incubation with LGALS3BP where three µg of total protein were used. Detection was by the chemiluminescent method. Results were representative of three experiments; ( B ) SDS-PAGE analysis of proteins of MBs and EVs. Ten µg of protein were applied per lane. Protein detection was with Coomassie R-250.

Article Snippet: The following antibodies were used: mouse anti-L1CAM (L1-11A) monoclonal (1:1000), mouse anti-CD63 monoclonal (1:500) (Invitrogen, Camarillo, CA, USA), mouse anti-CD9 monoclonal (1:5000), goat anti-human LGALS3BP polyclonal (1:2000) (R&D, Minneapolis, MN, USA), goat anti-Tsg101 polyclonal (1:200), goat anti-GRASP65 polyclonal (1:500), goat anti-calnexin polyclonal (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-annexin-I monoclonal (1:5000), mouse anti-human LAMP-1 monoclonal (1:500) (BD Biosciences Pharmingen, San Diego, CA, USA), mouse anti-EEA1 monoclonal (1:1000), mouse anti-GS28 monoclonal (1:250) (BD Transduction Lab, San Diego, CA, USA).

Techniques: Western Blot, Isolation, Incubation, SDS Page

Journal: Biomolecules

Article Title: Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

doi: 10.3390/biom5031741

Figure Lengend Snippet: List of proteins identified in EVs and MBs from OVMz cells using MALDI-TOF/TOF after SDS-PAGE separation using MALDI-TOF/TOF. Bands were excised from the gel shown in Figure 2 B.

Article Snippet: The following antibodies were used: mouse anti-L1CAM (L1-11A) monoclonal (1:1000), mouse anti-CD63 monoclonal (1:500) (Invitrogen, Camarillo, CA, USA), mouse anti-CD9 monoclonal (1:5000), goat anti-human LGALS3BP polyclonal (1:2000) (R&D, Minneapolis, MN, USA), goat anti-Tsg101 polyclonal (1:200), goat anti-GRASP65 polyclonal (1:500), goat anti-calnexin polyclonal (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-annexin-I monoclonal (1:5000), mouse anti-human LAMP-1 monoclonal (1:500) (BD Biosciences Pharmingen, San Diego, CA, USA), mouse anti-EEA1 monoclonal (1:1000), mouse anti-GS28 monoclonal (1:250) (BD Transduction Lab, San Diego, CA, USA).

Techniques: Sequencing

Journal: Biomolecules

Article Title: Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

doi: 10.3390/biom5031741

Figure Lengend Snippet: Deglycosylation of immunoprecipitated LGALS3BP. LGALS3BP was deglycosylated with Endo H, PNGase F, and sialidase from V. cholerae after immunoprecipitation from EVs (Ctr). The input EVs contained three µg of total protein. As control for the digestion the immunoprecipitate was incubated with the corresponding buffer (bufH for Endo H, bufF for PNGase F and bufS for sialidase). The controls of the immunoprecipitation without EVs ( w / o EVs) and without antibody ( w / o Ab) were also shown in the second panel. The blots are representative of two (Endo H) or four (PNGase F and sialidase) experiments. Immunoglobulin G bands are represented with *.

Article Snippet: The following antibodies were used: mouse anti-L1CAM (L1-11A) monoclonal (1:1000), mouse anti-CD63 monoclonal (1:500) (Invitrogen, Camarillo, CA, USA), mouse anti-CD9 monoclonal (1:5000), goat anti-human LGALS3BP polyclonal (1:2000) (R&D, Minneapolis, MN, USA), goat anti-Tsg101 polyclonal (1:200), goat anti-GRASP65 polyclonal (1:500), goat anti-calnexin polyclonal (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-annexin-I monoclonal (1:5000), mouse anti-human LAMP-1 monoclonal (1:500) (BD Biosciences Pharmingen, San Diego, CA, USA), mouse anti-EEA1 monoclonal (1:1000), mouse anti-GS28 monoclonal (1:250) (BD Transduction Lab, San Diego, CA, USA).

Techniques: Immunoprecipitation, Incubation