Journal: Scientific Reports

Article Title: The von Willebrand factor stamps plasmatic extracellular vesicles from glioblastoma patients

doi: 10.1038/s41598-021-02254-7

Figure Lengend Snippet: VWF is transported in circulating EVs from GBM patients. ( A ) Equal volume of protein lysates from EV fractions, obtained with SEC followed by ultracentrifugation, and their corresponding plasma, from one GBM patient and one healthy donor, were separated by SDS-PAGE and analyzed by immunoblot for VWF, FCN3, and HSP70. Blots are representative of n = 3. ( B ) mRNA expression of von Willebrand factor (VWF) and ficolin-3 (FCN3) in healthy subjects and GBM patients estimated from The Cancer Genome Atlas (TCGA, n > 500 for GBM patients). ( C ) Anomalies currently reported in VWF and FCN3 genes. ( D ) Equal volume of protein lysates from EV fractions (3 GBM and 3 Healthy) were separated by SDS-PAGE and analyzed by immunoblot for VWF, GM130 (putative intracellular membrane protein contaminant), and Apolipoprotein A1 (APOA1, plasmatic protein). Healthy donor plasma and total cell lysate from GBM cells serve as controls. Blots are representative of n > 7 individual samples. ( E ) Densitometric analysis was performed on 7 and 8 independent EV preparations from Healthy and GBM liquid biopsies, respectively. ( F ) Concentration of VWF in plasmatic EVs from healthy donors and GBM patients (n = 20). Mann–Whitney test, * p < 0.05, ** p < 0.01, *** p < 0.001. S.D. are shown in panel E and S.E.M. in panel F.

Article Snippet: EVs were separated and concentrated from 500 μl of plasma as described above and the pellet was directly lysed in boiling Laemmli for 10 min. Proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and blotted with the following antibodies, all diluted at 1/1000: CD9 (EXOAB-CD9A-1, SBI), CD63 (EXOAB-CD63A-1, SBI), VWF (sc-53466, Santa Cruz), FCN3 (CL7767AP, Cedarlane) , Apolipoprotein A1 (sc-376818, Santa Cruz), HSP70 (EXOAB-HSP70A, SBI), and GM130 (ab52649, Abcam) diluted at 1/1000.

Techniques: Clinical Proteomics, SDS Page, Western Blot, Expressing, Membrane, Concentration Assay, MANN-WHITNEY

Journal: Osteoarthritis and cartilage

Article Title: Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

doi: 10.1016/j.joca.2010.05.013

Figure Lengend Snippet: Fig. 1. Production of MGP by normal and OA chondrocytes. Proteins in RIPA buffer extracts from normal (NC) and osteoarthritic (OA) chondrocytes were Western blotted with (A) a polyclonal N-terminal human MGP peptide antibody which recognizes human MGP independently of its g-carboxylation status, (B) conformational specific cMGP peptide antibody which recognizes the mature fully g-carboxylated form of MGP in the presence of Caþþ, and (C) the ucMGP antibody which recognizes none or under-g-carboxylated human MGP. Equal amounts of total protein were added in each lane. Lane Bone in A contains human MGP protein partially purified from bone.

Article Snippet: Affinity purified goat anti-human fetuin polyclonal antibodies were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Western Blot

Journal: Osteoarthritis and cartilage

Article Title: Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

doi: 10.1016/j.joca.2010.05.013

Figure Lengend Snippet: Fig. 2. Presence of fetuin and a fetuineMGP complex in normal articular chondrocytes. Samples of cell lysates from normal articular chondrocytes were separated by 2-D gel electrophoresis as described in the Materials and methods. (A) Coomassie blue staining and (B) Western blot of the proteins shown in panel A with the mouse monoclonal anti- human fetuin antibodies. The characteristic shape of the stained fetuin is pointed out by an arrow in panel A and panel B shows that stained fetuin protein reacts with the anti-fetuin antibody (arrow). (C) Western blot of fetuin immunoprecipitated with mouse monoclonal fetuin antibodies from an RIPA buffer extract of normal chondrocytes and developed with mouse anti-cMGP antibody. The most heavy protein band labeled fetuinecMGP shows that the characteristically shaped fetuin molecule (arrows) reacts with the anti-cMGP antibody. The anti-cMGP antibody also recognized free cMGP that was seen on the blot (arrows, cMGP). The heavy chain of the anti-fetuin antibodies used to immunoprecipitate fetuin is recognized by the secondary monoclonal horseradish conjugated goat anti-mouse antibodies (labeled HIgG).

Article Snippet: Affinity purified goat anti-human fetuin polyclonal antibodies were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Nucleic Acid Electrophoresis, Staining, Western Blot, Immunoprecipitation, Labeling

Journal: Osteoarthritis and cartilage

Article Title: Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

doi: 10.1016/j.joca.2010.05.013

Figure Lengend Snippet: Fig. 3. MGP and fetuin in vesicles from normal and OA chondrocytes. Vesicles from normal (N) and osteoarthritic (OA) serum-free cultures (24 or 48 h serum-free as indicated) were isolated by centrifugation as described in Materials and methods. (A) Electron microscopic images of matrix vesicles isolated from OA chondrocyte cell cultures. The bars represent a distance of 300 nm. Two panels are shown in order to demonstrate the variety of vesicles. (B) Silver stained proteins present in the vesicles (V) from normal (lane V-N) and osteoarthritic (V-OA) cells. Equal amounts of total protein were loaded in each lane. (C) Western blots with the anti-cMGP antibody of the proteins shown in panel B. This conformational specific antibody recognizes only the mature fully g-carboxylated MGP in the matrix vesicles shed from normal chondrocytes. (D) and (E) Western blots of the proteins in vesicles isolated at 24 and 48 h from cultured cells, respectively, blotted with anti-fetuin antibody. Equal amounts of total proteins were loaded in each lane in panels D and E. (F) Coomassie blue stained proteins on the PVDF membrane used for fetuin Western blotting shown in panel E.

Article Snippet: Affinity purified goat anti-human fetuin polyclonal antibodies were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Isolation, Centrifugation, Staining, Western Blot, Cell Culture, Membrane

Journal: Osteoarthritis and cartilage

Article Title: Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

doi: 10.1016/j.joca.2010.05.013

Figure Lengend Snippet: Fig. 4. Presence of the cMGPefetuin complex in vesicles from normal chondrocytes. An RIPA buffer extract of vesicles isolated from normal chondrocytes was immunoprecip- itated with affinity purified goat anti-human fetuin antibody. The immune-precipitated proteins on SepharoseeProtein-A/G beads were separated in 2-D-SDS-PAGE gels and Western blotted with the mouse monoclonal anti-cMGP antibody. The characteristic shape of the fetuinecMGP complex was seen as the high molecular weight band (arrows) and free cMGP was also seen (low molecular weight band).

Article Snippet: Affinity purified goat anti-human fetuin polyclonal antibodies were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Isolation, SDS Page, Western Blot, High Molecular Weight, Molecular Weight

Journal: Osteoarthritis and cartilage

Article Title: Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

doi: 10.1016/j.joca.2010.05.013

Figure Lengend Snippet: Fig. 5. Immunolocalization of fetuin in human articular cartilage. Sections from human knee articular cartilage were processed for confocal microscopy as described in detail in Materials and methods. The section was reacted with a highly specific monoclonal rat recombinant anti-human fetuin peptide antibody followed by visualization on the immune complexes with a donkey rhodamine conjugated anti-mouse antibody. Panel A shows fetuin to be present in several lacuna. Panel B is an enlarged image to examine intra-cellular details of the rhodamine stained spots.

Article Snippet: Affinity purified goat anti-human fetuin polyclonal antibodies were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Confocal Microscopy, Recombinant, Staining

Journal: Osteoarthritis and cartilage

Article Title: Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

doi: 10.1016/j.joca.2010.05.013

Figure Lengend Snippet: Fig. 6. Biotin-labeled fetuin binding and uptake by cultured human chondrocytes. Human chondrocytes were depleted of endogenous fetuin as described in Materials and methods and incubated at 4 or 37C in serum-free medium containing biotin-labeled human fetuin. (A) Cells incubated with biotin-labeled human fetuin for 30 min at 4C. (B) Cells incubated with biotin-labeled fetuin for 30 min at 4C, followed by incubation at 37C for an additional 30 min; (C) same experiment as in (B) except that unlabeled fetuin was used in the incubations. Rhodamine epifluorescence images were obtained using a Zeiss Axioskop equipped with a digital camera and Axovision imaging software as described in Materials and methods.

Article Snippet: Affinity purified goat anti-human fetuin polyclonal antibodies were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Labeling, Binding Assay, Cell Culture, Incubation, Imaging, Software