Journal: International Journal of Molecular Sciences

Article Title: Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells

doi: 10.3390/ijms22083993

Figure Lengend Snippet: Co-localization of AnxA6, AnxA2, or fetuin-A (FetuA) with TNAP in hFOB 1.19 ( A , C , E ) and Saos-2 ( B , D , F ) cells in resting conditions (R) or after seven-day stimulation with AA and β-GP (S). The cells were incubated with appropriate antibodies: mouse monoclonal anti-AnxA6 ( A , B ), mouse monoclonal anti-AnxA2 ( C , D ), or mouse monoclonal anti-FetuA ( E , F ), all followed by goat anti-mouse IgG-FITC (green); rabbit polyclonal anti-TNAP followed by goat anti-rabbit IgG-TRITC (red) and DAPI for nuclei (blue) and observed under an Axio Observer.Z1 FM (Carl Zeiss, Poznan, Poland) with Phase contrast and appropriate fluorescent filters, magnification 630 x. Arrowheads indicate protein accumulation in vesicular and/or cluster structures. The yellow color and arrows on the merge images indicate AnxA6 ( A , B ), AnxA2 ( C , D ), or FetuA ( E , F ) co-localization with TNAP. Results of a typical experiment are presented.

Article Snippet: The membranes were then incubated with mouse monoclonal anti-annexin A6 (AnxA6; 1:1000, v/v ; BD Transduction Laboratories, Warsaw, Poland), mouse monoclonal anti-annexin A2 (AnxA2; 1:1000, v/v ; BD Transduction Laboratories, Warsaw, Poland), mouse monoclonal anti-fetuin-A (FetuA; 1:500, v/v ; Abcam, Cambridge, UK), rabbit polyclonal anti-TNAP (TNAP; 1:1000, v/v ; Abcam, Cambridge, UK), or mouse monoclonal anti-actin 1 (Actin; 1:2000, v/v ; Abcam, Cambridge, UK) primary antibodies prepared in 3% ( w/v ) milk in TBS supplemented with 0.05% ( v/v ) Tween-20 (TTBS), at 4 °C overnight.

Techniques: Incubation

Journal: Communications Medicine

Article Title: Spatial omics-based machine learning algorithms for the early detection of hepatocellular carcinoma

doi: 10.1038/s43856-024-00677-7

Figure Lengend Snippet: a Slides are spotted with 16 antibodies (top to bottom in columns) to the following antibodies: (1) anti-Alpha 1 Antitrypsin, (2) anti- Alpha 1B-Glycopotein, (3) anti-alpha 1-Acid Glycoprotein, (4) anti-Alpha-2-Macroglobulin, (5) anti-Angiotensinogen II/III, (6) anti-Apolipoprotein D, (7) anti-Apolipoprotein H (ApoH), (8) anti-Ceruloplasmin, (9) anti-Clusterin, 10) anti-Fetuin, 11) anti-Haptoglobin, 12) anti-Hemopexin, (13) anti histidine-proline rich glycoprotein; (14) anti-IgG, (15) anti-Transferrin; (16) anti-Vitamin D Binding Protein. B-D) Example of GlycoTyper data for the 16 captured proteins for N-glycan at 2539.881 ( b , e ), N-glycan at 2174.654 ( c , f ) and N-glycan at 1809.639 ( d , g ). b – d are from patients with HCC, while e – g are from patient with cirrhosis. h Bar graph with individual data points showing the mean (with SD) level of N-glycan at 2539.907 on haptoglobin; i Bar graph with individual data points showing the mean (with SD) level of N-glycan at 1663.581 on transferrin. j , k Similar analysis on angiotensinogen ( j ) hemopexin ( k ). l ROC curves for Model G, AFP and AFP-L3 of discriminant ability to classify all or early-stage HCC from cirrhosis. Where statistical difference exists, the p value is provided and error bars indicate the standard deviation.

Article Snippet: Mouse anti-Human Alpha 1B-Glycopotein (#MAB7757), Goat anti-Human alpha 1-Acid Glycoprotein (#AF3694), Mouse anti-Human Apolipoprotein H (#MAB5087), Mouse anti-Human Fetuin A/AHSG (#MAB1184), Mouse anti-Human HPRG (#MAB1869), Mouse anti-Human Vitamin D Binding Protein (#MAB3778) antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Glycoproteomics, Standard Deviation

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