Endoglin

Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100 (v/v), 1% IGEPAL (v/v), 0.1% SDS), and protein concentration was measured with BCA test (Thermo Fisher Scientific). Subsequently, lysates were supplemented with Laemmli buffer [93 (link)], boiled at 95 °C for 10 min prior to separation on 10% SDS–PAGE gels. Proteins were transferred onto nitrocellulose membranes (neoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg, Germany). Membranes were blocked in 5% w/v BSA (Sigma-Aldrich) in TBST and then incubated with primary antibodies overnight at 4 °C. The next day, membranes were incubated with goat-α-rabbit-HRP, goat-α-mouse-HRP (1:10,000, Dianova, Hamburg, Germany), or mouse-α-goat (1:10,000, Santa Cruz Biotechnology), prior to detection with WesternBright Quantum ECL HRP substrate (advansta, Menlo Park, USA) using a Fusion-FX7 (Vilber Lourmat, Eberhardzell, Germany). Primary antibodies used were Endoglin (AF 1097, R&D systems), Caveolin-1 XP (3267, Cell Signaling Technology), EEA1 (610456, BD Transduction Laboratories), pSMAD1/5 (9516, Cell Signaling Technology), and GAPDH (2118, Cell Signaling Technology). The concentration of primary antibodies was 1:1000 in 3% BSA.