Anti yap antibody

For Western blot analyses, proteins were extracted in buffer containing 8 M Urea, 100 mM Tris–HCl pH 8. Briefly, cell lysates (50 μg) were separated by SDS–PAGE using a precast polyacrylamide gel with a 4% to 12% gradient (Invitrogen). After the electrophoretic run, proteins were transferred onto a 0.22 μm nitrocellulose membrane, for dot blot we spotted the protein mix directly to nitrocellulose membrane (Amersham Protran, GE Healthcare) in wet conditions. The assembled sandwich was loaded in a Trans‐Blot Cell (Bio‐Rad) and immersed in 1× cold Tris‐Glycine transfer buffer with the addition of 20% methanol. The transfer was allowed overnight at constant voltage (30 V). Correct protein transfer was verified staining the membrane with Ponceau red (Sigma‐Aldrich) for few seconds. After washing the membrane with Tris‐buffered Saline‐Tween 20 (TBST, 1× TBS with 0.1% Tween‐20), non‐specific binding of antibodies was blocked by adding 5% low‐fat dry milk in TBST for 1 h at room temperature. The antibody used are anti-LATS1 (Cell Signaling, 1:1000, rabbit), anti-LATS2 (Cell Signaling, 1:1000, rabbit), anti-BACE2 (Sigma Prestige, 1:250, rabbit) and anti-OC (Merck, 1:1000, Rabbit), anti-YAP-antibodies (Santa Cruz, 1:200, mouse), anti-p-YAP (Ser127) (Cell Signaling, 1:1000, Rabbit).