Immunoblotting and Immunohistochemical Analysis

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.