Bca assay

BCa cells/tissues were harvested in RIPA buffer and protein content was determined via BCA Assays (Bio-Rad). Lysates were resolved via SDS-PAGE electrophoresis and proteins semi-dry transferred to nitrocellulose membranes (Invitrogen). Membranes were blocked and labeled with antibodies targeting WNT11, CTNND1, CyclinD1, β-catenin and anti-MMP7 (all purchased from Abcam) and secondary HRP-conjugated antibodies (Cell Signaling Technologies). GAPDH was probed as a loading control. Proteins were visualized using ECL (Millipore).

Protein concentrations were quantified by BCA assays (BIO-RAD). For western blots, proteins were denatured in 1 × RIPA buffer (Life Technologies). 2–5 μg of total protein were loaded onto pre-cast gels (10 or 12% MINI-PROTEAN TGX^® 12 by 15 or 50 μL well pre-casted gel, respectively; BIO-RAD). Separated proteins were transferred to nitrocellulose using the Trans-Blot^® Turbo Transfer System (BIO-RAD). Membranes were blocked in 5% milk in TBS-T for 1 h at room temperature. Primary antibodies were incubated overnight in 5% milk in TBS-T at 4 °C. Secondary antibodies were incubated in 5% milk in TBS-T for 1 h at room temperature. Blots were then washed in 1 × TBS-T three times. For visualization, membranes were treated with SuperSignal™ West Femto Maximum Sensitivity Substrate for 60 s (Thermo Scientific). Blots were then exposed to film and developed for ~ 1 s to ~ 5 min depending on band intensity.

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.