Western Blot Analysis of Protein Expression

Total protein was extracted 72 h after the transfection using RIPA Lysis Buffer (Beyotime Institute of Biotechnology,). The proteins were separated by SDS-polyacrylamide gel (12%) and were transferred to polyvinylidenedifluoride membranes (0.2 µm pore size) (EMD Millipore, Bellerica, MA, USA) and were then detected with a rabbit anti-p21 polyclonal antibody (sc-471, 1:600; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), a rabbit anti-Cdk1 monoclonal antibody (ab32384, 1:1,000; Abcam, Cambridge, MA, USA) (used to detect dephospho-Cdk1 (Tyr15) which refers to active Cdk1 signalling pathways) (21 (link),22 (link)), a mouse anti-Gas2 monoclonal antibody (M01, 1:1,000; Abnova, Taipei, Taiwan) and a mouse anti-α-tubulin monoclonal antibody (T5168, 1:5,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG or anti-mouse IgG; Beyotime Institute of Biotechnology,) were diluted 3,000-fold, and the signals were detected by an enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.).