Anti p lats1

Protein samples were diluted 1:5 with protein loading buffer (6 × Transgen Biotech, Beijing, China) and heated at 95 °C for 5 min. Protein extracts were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes, and blocked with 5% nonfat dry milk in TBST. The membranes were incubated with primary antibodies at 4 °C overnight and with the horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 1 hour. Protein bands were visualized by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA) and imaged by a FluorChem M gel documentation system (ProteinSimple, San Jose, CA). The primary antibodies (anti-YAP, anti-p-YAP, anti-LATS1, anti-p-LATS1, anti-MYPT1, anti-p-MYPT1, anti-CREB, anti-p-CREB, anti-GAPDH, anti-α-tubulin, anti-histone H3) and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). CTGF antibody was obtained from Sigma-Aldrich (St Louis, MO, USA).

Total proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China), and electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in Quick Block (Beyotime, Shanghai, China) for 15 min, then incubated with specific primary antibodies (1:1000) at 4°C overnight. Anti-PAX5 (Biorbyt, Cambridge, UK), anti-slug, anti-E-cadherin, anti-vimentin, anti-p-LATS1, anti-LATS1, anti-p-YAP, and anti-YAP (Cell Signaling Technology, USA) were used. Membranes were incubated in secondary antibodies at room temperature for 1 hour. TBST was used to wash unbound antibodies. An enhanced chemiluminescence detection system was used to detect target proteins. GAPDH and β-actin were internal controls.