Anti phospho fak

Protein extraction and immunoblot analysis were performed using a modified Laemmli sample buffer (125 mM Tris-HCl, pH 6.8 buffer containing 2% SDS and 20% glycerol) in the presence of protease and phosphatase inhibitors (Roche, Vilvoorde, Belgium). Lysates were separated by SDS-PAGE under reducing conditions, transferred to a nitrocellulose or PVDF membrane, and analyzed by immunoblotting. Primary antibodies used were anti-LC3 and anti-BNIP3 from Cell Signaling (Leiden, The Netherlands), anti-VE-cadherin from R&D Systems (Abingdon, UK), anti-phospho-FAK from BD-Transduction Laboratories (Ermebodegem, Belgium), anti-ZO-1 from Invitrogen, anti-Vinculin and anti-Integrin from Millipore (Merck, Overijse, Belgium), anti-Actin, anti-Vinculin and anti-Vimentin from Sigma (Diegem, Belgium). Equal loading was verified by actin immunostaining. Appropriate secondary antibodies were from Thermo Scientific (Erembodegem, Belgium). The LICOR Odyssey System (Westburg, Leusden, The Netherlands) was used for western blot detection according to the manufacturer's instructions. Quantifications were performed using the Odyssey System software. Representative blots of at least three independent experiments are shown.