Protein preparation and immunoblotting
Protein extracts were prepared as previously described (Kenney and Rowitch, 2000). Protein content was determined by using the Bio-Rad protein assay. 50-75 μg of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8-10% polyacrylamide gels and then transferred in 20% methanol buffer at 4°C to Immobilon polyvinylidene difluoride (Millipore) membranes. Immunoblotting was carried out according to standard methods. Antibodies used for western blotting were: YAP1 (Abcam), phospho-Akt (Ser473), phospho-Cdk1(Tyr15), phospho-Chk2 (Thr68), total Akt and cleaved caspase 3 (Cell Signaling), phospho-ATM (Ser1981) (Rockland), Chk2 (Millipore), IGF2 and β-tubulin (Sigma), VEGF, Cyclin D2 and Cyclin B1 (Santa Cruz). Donkey anti-mouse HRP-linked secondary was from Jackson Research Laboratories and goat anti-rabbit from Thermo Scientific. Peroxidase activity was detected using Amershams’s ECL reagents and exposing membranes to Kodak Biomax film.