Crk l

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).

Sp1 siRNA, miR-29b and their scrambles were introduced into K562 cells by Lipofectamine™ 2000 (Invitrogen) and the Western blotting was performed as previously described [20 (link), 26 (link), 27 (link)]. The antibodies used are: Sp1, DNMT3a, β-actin and ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA); ABL, phospho-ABL, caspase-3 and caspase-8 (Cell Signaling Technology, Danvers, MA); Parp, CRKL and p-CRKL (Millipore, Billerica, MA).