Fig. 4

Effect of ADAM inhibitors on VE-cadherin protein levels. a) Endothelial cells pre-treated 30 min before irradiation (4 Gy) with vehicle alone (DMSO, 0.1%) or with inhibitors of ADAM10 (GI254023X, 10 μM), and ADAM17 (GW280264X, 10 μM) were lyzed and subjected to immunoblot analysis and quantitative evaluation (n ≥ 3; β-actin served as loading control). b) Endothelial cells were in the presence of absence of the ADAM10-inihibitor GI254023X (10 μM) treated with APMA (100 ng/ml; for 2 h only) or TNFα (100 ng/ml) and analyzed 24 h later as described in A (n ≥ 2). c) Quantification of the 35-kDa intracellular C-terminal fragment of VE-cadherin detected by immunoblot analysis as described in A but in the presence of a γ-secretase-I inhibitor (1 μM) in order to stabilize the proteolytic fragment (n ≥ 3). d) Quantification of the soluble 90-kDa N-terminal VE-cadherin fragment by ELISA. For this purpose, a total of 10⁶ cells in 3 ml medium were seeded into 8-cm²-dishes 24 h before and treated with GI254023X (10 μM) 30 min before irradiation (4 Gy). After 24 h, the cell culture supernatant was assayed and the amount of soluble VE-cadherin (ng) per 100,000 cells originally seeded was calculated (n ≥ 4). Exemplary immunoblots are shown (a–c). Data are shown as means ± standard deviations. Statistics: t-test, *p < 0.05, **p < 0.01, ***p < 0.001