recombinant human et  (R&D Systems)

Journal: The FASEB Journal

Article Title: Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes

doi: 10.1096/fj.201801319R

Figure Lengend Snippet: Neuronal exosomes contain ECE-1 and -2 activity. A) Aβ was measured by sandwich ELISA in exosomes isolated from SH-SY5Y-APP cells grown in serum-free medium (control, C). Levels of the peptide increased with PA treatment and decreased with DAPT (100 µM, 48 h). B) SH-SY5Y-APP exosomes contained specific PA-sensitive activity for ECE-1 (at pH 6.8) and ECE-2 (at pH 5.5), measured with a big ET-1 conversion assay. **P < 0.01, ***P < 0.001.

Article Snippet: EVs from SH-SY5Y cells were solubilized in 20 mM Tris-HCl (pH 7.4), containing 250 mM sucrose and 2.5% C 12 E 10 (polyoxyethylene-10-lauryl ether), and 2 µg protein was incubated with 100 nM recombinant human big ET-1 (Enzo Life Sciences, Farmingdale, NY, USA) in 50 µl of either 0.1 M sodium phosphate (pH 6.8) or 0.1 M sodium acetate buffer (pH 5.5) containing 0.5 M NaCl and protease inhibitor cocktail, without EDTA, supplemented with 1 µM thiorphan.

Techniques: Activity Assay, Sandwich ELISA, Isolation