Yap1 ep1674y

For IP experiments, HEK293FT cells were transfected with 500 ng each of YAP and nV5-TEAD4 cDNA constructs, and lysed in RIPA buffer (derived from 10x stock (Millipore, Billerica, MA); final concentration of components: 50 mM TRIS.HCl pH 7.2, 120 mM NaCl, 1% Nonidet P40 (v/v), 1 mM EDTA and 0.1% (v/v) SDS; supplemented with 2 mM sodium orthovanadate, 6 mg/ml sodium pyrophosphate and PhosSTOP and Protease Inhibitor Cocktail (both from Roche, Switzerland)) 48 hr afterwards. Lysates (250 µg) were then incubated with YAP1 antibody overnight under rotation at 4°C, followed by incubation with Dynabeads Protein G (Invitrogen, Carlsbad, CA) for 2 hr under rotation at 4°C. Immunoprecipitates were washed three times with RIPA buffer lacking SDS, eluted with Laemmli Sample Buffer (BioRad, Hercules, CA) by incubation at 95°C for 5 min and resolved by standard SDS-PAGE gel electrophoresis and Western Blotting. The following antibodies were used. For IP: YAP1 (EP1674Y; Abcam (United Kingdom),ab52771). For Western Blot analysis: YAP1 (D8H1X XP; Cell Signaling Technology (Danvers, MA), #14074) and V5 (Invitrogen (Carlsbad, CA), R96025) as primary antibodies; HRP-anti-rabbit (Cell Signaling Technology (Danvers, MA), #7074) and HRP anti-mouse ( GE Healthcare (United Kingdom), NA931V) as secondary antibodies.

Tumor tissue samples from all the patients were fixed in buffered 4% formalin, embedded in paraffin, and stained according to standard procedures. The immunohistochemistry followed standardized methods in the Hospital Universitario de Cruces Pathology Lab, Spain. We used β-catenin (14, Roche Tissue Diagnostics, Roche Ltd.), p53 (Bp53-11, Roche Tissue Diagnostics, Roche Ltd., Oro Valley, AZ, USA), YAP1 (EP1674Y, Abcam Ltd.), GAB1 (H7, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and OTX2 (1H12C4B5, Thermo Fisher Scientific, Waltham, MA, USA) as primary antibodies. Immunohistochemical staining was performed in an automated immunostainer (BenchMark ULTRA Slide Stainer IHC/ISH System, Roche Tissue Diagnostics, Ventana Medical Systems, Roche Ltd. Oro Valley, AZ, USA).