Anti α tubulin and anti β actin antibodies

Cells were lysed in RIPA buffer containing protease inhibitors on ice for 15 minutes. Protein samples were obtained by centrifugation at 13,000 rpm for 20 minutes. Equal amounts of proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were incubated at 4°C overnight with primary antibodies. Primary antibodies against the following molecules were purchased from Cell Signaling Technology (Beverly, MA): p53, RAD51, PTEN, AKT, phosphorylated AKT (Ser473), Src, and phosphorylated Src (Tyr416). Following antibodies were purchased from Santa Cruz Biotechnology: cyclin D1, cyclin E, cyclin A, Jab1, γH2AX, and p27. Anti-α-tubulin and anti-β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Antibody binding was detected using an enhanced chemiluminescence system according to the manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ). Anti-mouse and rabbit secondary antibodies were purchased from Thermo Scientific Inc. (Waltham, MA). The data was quantified by ImageJ software (National Institute of Health, Bethesda, MD) and normalized by α-tubulin or β-actin as an internal control.