Western Blot Analysis of YAP Expression

Western blots were carried out as previously described48 (link)49 (link). Briefly, 50–100 μg of protein from whole cell extracts from both tumor and non-neoplastic liver were re-suspended in 4X tris-glycine sample buffer, boiled at 95 °C for 5–10 min, and fractionated on a 4–20% tris-glycine gel (Invitrogen, Carlsbad, CA) for 90 min at 125–130 V. Proteins were transferred to PVDF membranes and then blocked with a solution of either 5% BSA or 3% BSA and 5% milk in tris-buffered saline with Tween 20 (TBST) before immunodetection with the following antibodies: anti-YAP (Cell Signaling #4912, Danvers, MA), and anti-GAPDH (Millipore #MAB374, Billerica, MA). Overnight primary antibody incubation was followed by washes (60 min at room temperature) in TBST before incubation in secondary antibody for 1 hour (horseradish peroxidase-conjugated goat anti-rabbit IgG, Cell Signaling; or goat anti-mouse IgG, Bio-Rad, Hercules, CA). After washing with TBST, proteins were visualized using the ECL detection system (NEN, Boston, MA). Coomassie gels were used to ensure equal protein loading. Band densities were analyzed using AlphaEase FC software version 4.1.0 (Alpha Innotech) to determine relative protein expression, and were then normalized to GAPDH band densities.