Hrp conjugated anti rabbit or anti mouse antibody

DOX was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). Caspase-9, Caspase-3, Bcl-2, Bax antibodies and secondary antibodies directed against rabbit or goat (HRP-conjugated anti-rabbit or anti-mouse antibody) were purchased from Proteintech Group (Chicago, IL, USA). ROS detection kit for flow cytometry was purchased from Enzo R Life Sciences (Farmingdale, NY, USA). Fluorometric TUNEL system was purchased from Promega (Madison, WI, USA). All chemicals and solvents were of analytical grade.

Cells were washed with PBS (pH 7.4), and incubated with 2× concentrated electrophoresis sample buffer (125 mM Tris-HCl, pH 6.8, 5% glycerol, 2% SDS, 1% β-mercaptoethanol) for 30 min on ice. Protein concentration was determined with Coomassie protein assay reagent using bovine serum albumin as a standard. Total protein (50–70 µg/lane) from the whole cell lysates was separated by 12% SDS-PAGE and proteins separated in the gel were transferred electrophoretically onto nitrocellulose membrane (Millipore Billerica, MA, USA). Antibodies against HDAC3, Ac-p53 (k382) (1∶1000) were obtained from Abcam (Cambridge, UK). Ac-p53 (k373) (1∶1000) was obtained from Upstate-Millipore (Massachusetts, USA). p53 and p21 (1∶200) were obtained from BD (Pharmingen, USA). PCNA, PRB, cyclin E, cyclin D1, CDK4, CDK2 and GAPDH antibodies (1∶500) and HRP-conjugated anti-rabbit or anti-mouse antibody (1∶2000) were obtained from Proteintech Group (Chicago, IL, USA). ECL (enhanced chemiluminescence) detection system (Bio-Rad) was used to visualize immunoreactive bands.