Gel transfer device

The western blot protocol followed was the same as.⁸ HUVECs were grown on FN-coated 6-well culture plates and washed twice with PBS++ (PBS + 0.5 MgCl₂ and 1 mM CaCl₂). For sample lysis, NP40 lysis buffer (50 mM TrisHCl, 100 mM NaCl, 10 mM MgCl₂, 1% NP40 and 10% glycerol, pH7.4) with 1:500 protease inhibitor was used. Protein samples were centrifuged at 14.000 xG at RT for 10 minutes and resuspended in SDS-sample buffer containing 4% β-mecapto-ethanol. Samples were boiled at 95°C for 3 minutes to denature proteins and separated on a 4–12% NuPage Bis-Tris gel (Invitrogen, NP0322BOX). Proteins were transferred using an iBlot² (link) Gel Transfer device (Invitrogen, IB21001) for 7 minutes to a nitrocellulose membrane (Invitrogen, IB23001). Then, membranes were blocked with a 5% milk solution in tris-buffered saline with Tween 20 (TBST) at RT for 30 minutes. Primary antibodies were incubated overnight at 4 degrees in TBST. Secondary HRP antibodies were incubated at RT for 1 hour, after which a Pierce ECL Western Blotting Substrate kit (Themo Scientific, 32106) was used according to manufacturer’s instructions. After each blocking and staining step, the membranes were washed with TBST 3x minutes. Western blots were developed using an Amersham imager 600.