Phospho rb p rb s780

Cell lysates were separated by SDS-PAGE, and the proteins were then transferred to PVDF membranes. The proteins were detected using antibodies against the following: RB (ab181616, 1:2000), cyclin D1 (ab40754, 1:1000), E2F1 (ab179445, 1:1000) (Abcam, Cambridge, UK), phospho-RB (p-RB) (S780) (#8180, 1:1000), PARP/cleaved PARP (#9542, 1:1000) (Cell Signaling Technology), and β-Actin (AC026, 1:100,000) (ABclonal, Boston, USA). Specific bands were visualized by ECL (Advansta, USA) and detected with an imaging system (Bio-Rad, USA).

Cell lysis was performed using RIPA buffer (Sigma) supplemented with phosphatase inhibitors (PhosSTOP™ EASYpak Phosphatase Inhibitors Cocktail, Roche) and protease inhibitors (cOmplete™ Protease Inhibitor Cocktail, Roche). Proteins were quantified and separated by SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) according to standard protocols. Membranes were immunoblotted with antibodies against p21 and p53 from Santa Cruz Biotechnology, and phospho‐Rb (pRBS780) from Cell Signaling. After incubation with the primary antibody overnight, membranes were washed and incubated with secondary HRP‐conjugated AffiniPure antibodies (Jackson ImmunoResearch) for 1 hr at room temperature and subsequently incubated with Enhanced Chemiluminescence Detection solution (Amersham). Membranes were placed on X‐ray films and processed using a Xograph Compact X4 automatic processor.