Western blots

Total cell lysates were prepared after washing cells with ice-cold phosphate-buffered saline (PBS), lysed in RIPA buffer (Sigma R0278) containing freshly added protease inhibitor (Roche #04693159001) and PhosSTOP phosphatase inhibitor cocktail (Roche #04906837001). The protein amount in cell lysates was quantified by BCA protein assay (ThermoFisher Scientific #23228). Equal amounts of protein extracts were boiled with 4x laemmli buffer, loaded in SDS-PAGE, and transferred to the PVDF membrane (Millipore #IPVH00010). The membranes were then blocked with 5% non-fat milk or BSA for 1 h at room temperature and incubated overnight at 4 °C with 1:1000 dilution of primary antibodies (YAP/TAZ: Cell Signaling Technology (CST) #8418; YAP: CST #14074; beta-tubulin: CST #86298; SKP2: Thermo Fisher Scientific #323300; vinculin: Santa Cruz Biotechnology #sc-73614). The membranes were later incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG antibodies (CST #7074 or CST #7072) for 1h at room temperature. After washing, the membranes were developed and captured by Amersham ECL prime western blotting detection reagent (GE Healthcare Life Sciences RPN2232) and Bio-Rad ChemiDoc^TM Imaging System.