Western Blot Analysis of Protein Expression

Cells were lysed in RIPA buffer containing a protease (TargetMol, CC0001) and phosphatase (TargetMol, CC0004). Protein concentrations were assessed using the BCA Protein Assay Kit (Pierce Biotechnology). Equal amounts of protein mixed with sample loading buffer (5X; Beyotime) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk or BSA in Tris-buffered saline Tween-20 (TBST), the membranes were incubated with primary antibodies overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The protein bands were visualized using a chemiluminescence kit (Pierce). GAPDH was used as a control for equal protein loading. The primary antibodies included anti-RDH5 (Abcam, ab200197), anti-GAPDH (Proteintech, 60004-1-1g), anti-YAP1 (Proteintech, 13584-1-AP), and anti P-YAP antibodies (CST, 13008), anti-mouse and anti-rabbit peroxidase-conjugated secondary antibodies were used 25 (link), 26 (link). All experiments were independently repeated at least three times.