Western Blot Analysis of Cell Signaling

Western blotting was performed as previously described [3 (link)]. Briefly, the cells were lysed with RIPA buffer (Sigma Aldrich) containing 150 mM NaCl, 1.0% Nonidet-P 40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8.0), a protease inhibitor cocktail, and PhoSTOP (Roche Molecular Biochemicals, Basel, Switzerland). All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against VE-cadherin, p-LKB1, and LKB1 were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Secondary antibodies (anti-rabbit IgG or anti-mouse IgG, 1:2000) were purchased from Cell Signaling Technology. Image densities were quantified using the National Institutes of Health ImageJ 1.41q software.