Anti phospho ser897 and anti phospho tyr772 antibodies

Cells were transfected with plasmids using Lipofectamine 3000, grown for about 12 h, serum-starved for ~12 h and treated with FBS for 15 min, in some cases in the presence of 5 μg/ml (200 nM) m-ephrinA1. Cell lysates were collected with lysis buffer (25 mM Tris-Cl, 0.5% TritonX-100, 20 mM NaCl, 2 mM EDTA and phosphatase and protease inhibitors (Roche Applied Science)). The lysates were centrifuged at 14,000 × g for 15 min at 4 °C and stored at −20 °C. BCA assays (Bio-Rad) were used to measure the protein concentrations of the samples. Lysates mixed with LDS sample buffer and reducing buffer were run on 3–8% NuPAGE^HNovex^HTris-Acetate mini gels (Invitrogen, CA) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in 1 × TBST. EphA2 expression was quantified using anti-EphA2 antibodies (Cell Signalling, MA). S897 and Y772 phosphorylation levels were quantified using anti-phospho-Ser897 and anti-phospho-Tyr772 antibodies (Cell Signaling, MA) followed by an anti-rabbit HRP conjugated antibody (Promega, WI). Nitrocellulose membranes were incubated for 2 min with Amersham ECL Plus Western Blotting Detection Reagent (GE Health Care Life Sciences, PA) and exposed from 1 to 60 s to capture images with the ChemiDoc imaging system (Bio-Rad, CA). Uncropped images of all Western blots presented in the study are shown in Supplementary Figures 13-18.