Rhadam17

Catalytic activity of the rhADAM17 (100 ng/ml, purchased from R&D Systems, MN, USA) was measured by hydrolytic processing of Fluorogenic Peptide Substrate III Mca-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH2 (20 µM, R&D Systems, MN, USA) followed by monitoring the increasing fluorescence intensity at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 10, 30 and 120 min at 37 °C with a Synergy H4 plate reader (BioTek, VT, USA). All reactions were performed in a fluorogenic buffer containing 25 mM Tris, pH 9.5, 150 mM NaCl, 2.5 µM ZnCL₂ and 0.005% Brij-35. For the rhADAM17 in vitro CA IX cleavage activity evaluation, the FL-CA IX-C-SBP protein was immobilised to High-Capacity Streptavidin Agarose (Thermo Fisher Scientific, MA, USA), washed and incubated with recombinant ADAM17 (dissolved at a concentration of 100 ng/ml in 25 mM Tris, pH 9.5, containing 2.5 µM ZnCL₂ and 0.005% Brij) for 3 h at 37 °C. Thereafter, the supernatant was analysed by ELISA as described below.

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).