4.6. Western Blot Analysis

Cells were harvested and the pellets were incubated in RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitors for 30 min on ice. Subsequently, at least 40 μg of extracted proteins were electrophoresed through 4–20% poly-acrylamide gels. After transferring the proteins to PVDF membranes using a TransBlot Turbo system (Bio-Rad, Hercules, CA, USA), the membranes were blocked with 5% non-fat milk in PBS with 0.1% TWEEN^® 20 for 1 h and then incubated separately with primary antibodies at 4 °C overnight. Membranes were then incubated with HRP-conjugated secondary antibody and stained with an enhanced chemiluminescence kit (Clarity Western ECL Substrate, Bio-Rad). The signals were acquired by ChemiDoc Imaging System XRS + (Bio-Rad). In order to detect the expression levels of GSK-3α/β, its signaling-related proteins were used: GSK-3α/β (CST, Cat#5676), phospho-GSK-3α/β (CST, Cat#5558) and rabbit monoclonal anti-phospho-β-Catenin (Ser675) (CST, Cat#8814). To detect the expression levels of cell cycle-related proteins, the following antibodies from Cell Signaling Technology (Boston, MA, USA), Santa Cruz Biotechnology (Dallas, TX, USA) and Abcam (Cambridge, UK) were used: Cdk1/Cdk2 (sc-53219), Cyclin B1 (CST, Cat#4138) HistoneH3 (CST, Cat#4499) and phospho-HistoneH3 (Ser10) (CST, Cat#9701). Furthermore, to evaluate the expression levels of the mitotic spindle check-point proteins MAD2 (sc-374131), the anti-gamma-H2A.X (ab-11174) and p21 Waf1/Cip1 (12D1) (CST, Cat#2947) antibodies were used. Finally, to detect autophagy marker expression and autophagy signaling related proteins the following antibodies were employed: LC3A/B (CST, Cat#4108), SQSTM1/p62 (CST, Cat#5114), AMPK (CST, Cat#2532), phospho-AMPK (Thr172) (CST, Cat#2535), mTOR (sc-517464), phospho-mTOR (ser2448) (ab-1093), phospho-Raptor (Ser792) (CST, Cat#2083) and phospho-ULK (ser757) (CST, Cat#14202). Mouse monoclonal anti-β-Actin (CST, Cat #4970) antibody was used as a loading control.