Anti cdk2

Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-RB (GeneTex, Irvine, CA, USA), anti-phospho-Akt(Ser473) (Cell Signaling), anti-Akt (Cell Signaling), anti-phospho-Erk1/2(Thr202/Tyr204) (Cell Signaling), anti-Erk1/2 (Cell Signaling), anti-TOP2A (Cell Signaling), anti-CCND1 (Cell Signaling), anti-γH2AX (Cell Signaling), anti-CDK2 (GeneTex), anti-RAD51 (GeneTex), anti-CDK4 (Cell Signaling), and anti-GAPDH (GeneTex) antibodies followed by horseradish peroxidase-conjugated secondary antibodies and visualization using an enhanced chemiluminescence detection system. Immunohistochemical (IHC) sample preparation and staining with anti-GFAP (Genetex), anti-phospho-RB (Cell Signaling), anti-Ki-67 (Cell Signaling), and anti-γH2AX (Cell Signaling) antibodies were carried out as previously described [48 (link)]. Uncropped Western blot images are provided in Figure S5.

Total cellular proteins were extracted by lysis of the harvested cells with the protein extraction buffer at 4 °C for 1 h, followed by denaturation at 95 °C for 5 min, and then separated by regular sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) according to the procedure reported formerly [24 (link)]. Next, the proteins were transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA), after which the membrane was probed for the proteins of interest with specific antibodies. Finally, the immunoreactive bands were detected with an enhanced chemiluminescence (ECL) detection system (Amersham Life Sciences Inc., Arlington Heights, IL, USA). The expression of GAPDH was considered as a loading control. Primary and secondary antibodies used in this study includes: anti-LC3, anti-p62, anti-Beclin-1, anti-Bax, anti-Atg5, and anti-cyclin B (Cell Signaling Technology, Beverly, MA, USA); anti-p53, anti-PARP, anti-Caspase 3, anti-pro-Caspase 3, anti-cyclin A, and anti-GADPH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-p27, anti-CDK2, anti-mouse IgG, and anti-rabbit IgG (GeneTex, Irvine, CA, USA).