Timp3

Cells were recovered from culture, lysed and processed for Western blotting using standard protocols (antibodies are listed in Supplementary Table I). For semi-quantitative RT-PCR, cells were processed using Trizol reagent (Life Technologies) and total RNA was purified by ethanol precipitation (primers are listed in Supplementary Table II). For Notch-reporter assays, HBMECs were transfected with the Notch-reporter construct and Renilla luciferase as loading control (9 (link)). Reporter-transfected cells were treated with purified fibulin-3 for 16 h and processed to quantify luciferase activity. To measure alpha-secretase (ADAM10/17) activity, HBMECs 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. Lysates (50 μg total protein) were incubated with a fluorogenic ADAM10/17 substrate peptide (TACE substrate III, 10 μM; R&D systems, Minneapolis, MN) and development of fluorescence was followed with a microplate reader as recommended by the peptide manufacturer. Cultures were treated for 1 to 16 h with purified fibulin-3 (300 ng/ml), purified TIMP3 (1 μg/ml, Sigma-Aldrich), gamma-secretase inhibitor DAPT (25 μM, Tocris Bioscience, Bristol UK) and alpha-secretase inhibitor TAPI-2 (1 μM, Cayman Chemical, Ann Arbor MI)