Journal: eLife

Article Title: Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

doi: 10.7554/eLife.99172

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-ADAM10 11G2 , , Diaclone: #857.800.000 , WB (1/1000).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Marker, Sequencing, Purification, Transduction, Control, Software, Staining

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: The ADAM10 multimodular structure and its different forms. ( A ) Furin/PC7 proteases cleave Pro-ADAM10, the inactive protein containing a pro-domain (85 kDa), to activate the enzyme during the transit through the Golgi compartment. The full-length active form (ADAM10 FL, 60–65 kDa) is then directed to the cell membrane where it works as sheddase. ADAM10 itself can be subject to ectodomain shedding, a process that leads to the formation of the soluble (sADAM10, 50–55 kDa) and membrane-anchored C-terminal domain (ADAM10 CTF, 10 kDa). Antibody binding sites used in this study to recognize ADAM10 protein. ( B ) Western blotting membranes using the ProSci 2051 antibodies (detection of the C-terminal region) in platelets and Abcam 39153 (detection of the N-terminal region) in plasma and CSF. MK: standard marker. ( C ) Western Blot analysis of 10 μg and 20 μg aliquots of plasma obtained from two control subjects (P1 and P2). Both ADAM10 antibodies recognize a band with an apparent molecular weight of 50 kDa.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Membrane, Binding Assay, Western Blot, Clinical Proteomics, Marker, Control, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: Sociodemographic, clinical variables and neuropsychological evaluations of subjects in the groups.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: Western blotting assays and ADAM10 protein quantification in CSF and plasma. ( A ) Western blotting and quantification of CSF and ( B ) plasma samples from controls (cognitively healthy), aMCI and mild AD participants probed against anti-N-terminal ADAM10 antibodies, which recognized a 50 kDa form of the protein. Serum albumin (66 kDa) from all participants was used as load control for the quantifications. The last lanes of plasma membranes were loaded with young control samples (red squares). * p = 0 .02; ** p = 0.01 (control vs mild AD). One-Way ANOVA and Mann-Whitney test. Graph Pad Prism 8.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Western Blot, Clinical Proteomics, Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: Analysis of ADAM10 activity in SH-5YSY neuroblastoma cells, platelets or CSF samples. ( A ) Western blotting analyses of subcellular fractions of SH-SY5Y human neuroblastoma cells; samples were probed for ADAM10 (immature form 85 kDA, mature form 65 kDa), GAPDH (37kDa) and Histone H3 (17kDa), Calnexin (90 kDa) and APP (106-110 kDa). ( B ) Graph reporting ADAM10 activity monitored for 60 min of reaction in the same fractions described in ( A ). The activity of ADAM10 was evaluated using a fluorogenic peptide as described in methods. ( C ) ADAM10 activity in CSF (rightmost graph) and in plasma (leftmost graph) of control cognitively healthy (control), amnestic mild cognitive impairment (aMCI) and mild Alzheimer’s disease (Mild AD) groups. The ADAM10 activity was also analyzed in SH-5YSY neuroblastoma cells, platelet samples and recombinant ADAM10. A specific inhibitor for ADAM10 GI254023X was incubated with platelets before the addition of substrate to demonstrate that the majority of the activity in samples was due to ADAM10 (* p < 0.0001). Two-Way ANOVA and Kruskal-Wallis test. Graph Pad Prism 8.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Activity Assay, Western Blot, Clinical Proteomics, Control, Recombinant, Incubation