Immunoblotting Protocol for Protein Analysis

Immunoblotting was performed according to a previously described method [27 (link), 28 (link)]. Briefly, after incubation in indicated concentrations of inhibitor, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation lysis buffer. The protein content of lysates was determined with a kit (Bio-Rad, Hercules, CA, USA) and proteins were resolved by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) that was incubated with primary antibodies at the appropriate dilution for 1 h. The blots were then probed with the secondary antibodies and developed with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Little Chalfont, UK). Antibodies against the following proteins were used: phosphorylated (p-)ABL, p-Crk-L, cleaved caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and p-Aurora, p-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (all from Cell Signaling Technology); Crk-L (Millipore); Aurora A (GeneTex, Irvine, CA, USA); and ABL, c-Myc, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Three independent experiments were performed. Protein band intensity was evaluated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).