Transfected cells were lysed and processed for semiquantitative RT-PCR or Western blotting using standard protocols16 (link) (antibodies and primers are listed in Suppl. Tables S-I and S-II ). Cultures were treated overnight with the gamma-secretase inhibitor DAPT (25 μM, Tocris Bioscience, Bristol UK) or the TACE inhibitor TAPI-2 (1 μM, Cayman Chemical, Ann Arbor MI). Cells were also treated with TNFα (10 ng/ml, Peprotech, Rocky Hill NJ), fibulin-3 (300 ng/ml, Origene, Rockville MD), or the TNFα inhibitor C87 (10 μM, Tocris) for 1 to 24 h before collection. For reporter assays a plasmid carrying Renilla luciferase was co-transfected as loading control.
To measure TACE activity, cells were lysed in 50 mM Tricine buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2, 1 mM ZnCl2 and 0.1% Triton X-100. Clarified lysates (50 μg total protein) were incubated with a fluorogenic ADAM10/17 substrate peptide (TACE substrate III) as described18 (link).
To measure TACE activity, cells were lysed in 50 mM Tricine buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2, 1 mM ZnCl2 and 0.1% Triton X-100. Clarified lysates (50 μg total protein) were incubated with a fluorogenic ADAM10/17 substrate peptide (TACE substrate III) as described18 (link).
