ADAM10 and ADAM17 Cleavage Assay

Substrates rhN-cadherin extracellular domain (R&D Systems), GST protein (Abnova), rhTNFα (LifeSpan BioSciences) and rhRELT extracellular domain (LifeSpan BioSciences) Proteases were rhADAM10 and rhADAM17 (R&D Systems). Substrates (0.25 μg) and proteinases (0.25) μg with or without GI254023X in cleavage assay buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl₂ and 1 mM ZnCl₂, pH 7.5)^{39 (link)} were incubated at 37 °C overnight^{3 (link)}. Immunoblots were performed as listed above. Antibodies were, sheep anti-N-cadherin polyclonal antibody (0.2 μg/ml; R&D Systems), mouse anti-RELT monoclonal antibody (1:1000; Thermo Fisher Scientific) and rabbit anti-GST polyclonal antisera (1:1000 Cell Signaling Technology). Secondary antibodies were, anti-sheep IgG, HRP-conjugated (1:4000; Thermo Fisher Scientific), anti-mouse IgG, HRP-conjugated (1:3000; Cell Signaling Technology) and anti-rabbit IgG, HRP-conjugated secondary antibodies (1:3000; Bio-Rad).