Aurora a kinase

Immunoprecipitation was performed as described previously at 4°C, unless otherwise specified [62 (link)]. Approximately 10⁷ cells in 1 ml of cold 1× RIPA buffer containing protease inhibitors (Roche Diagnostics, Basel, Switzerland) were incubated on ice for 30 min with occasional mixing. Cell lysates were centrifuged at 12,000 × g for 10 min. The supernatant was collected, mixed with primary antibody [calgranulin B (Santa Cruz Biotechnology) or aurora A kinase (Abcam)], and incubated for 2 h with rocking. Prepared protein G Sepharose beads (100 μl; GE Healthcare Life Sciences) were added, incubated on ice for 1 h with rocking and centrifuged at 10,000 × g for 30 s. The supernatant was removed and protein G Sepharose beads were washed five times with 1 ml of cold 1× RIPA to minimize background. Next, 100 μl of 2× SDS sample buffer was added to the bead pellet and heated to 100°C for 10 min. After boiling, immunoprecipitates were centrifuged at 10,000 × g for 5 min, and the supernatant was collected for western blot analysis.

Western blot analysis was performed as described previously [30 (link)]. Briefly, protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) and blocked in 5% non-fat dry milk for 1 h at room temperature. Membranes were incubated with primary antibody against calgranulin B (Santa Cruz Biotechnology, Dallas, TX, USA), aurora A kinase (Abcam, Cambridge, MA, USA), c-Caspase3 (Cell signaling, Massachusetts, USA), p-AKT (Cell signaling), p-ERK (Cell signaling), p-JNK (Cell signaling), NF-κB (Cell signaling), p53 (Cell signaling), p38 (ABcam), β-catenin (ABcam), E-cadherin (Cell signaling), and β-actin (Santa Cruz Biotechnology) or actin (Sigma-Aldrich, St. Louis, MO, USA). Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Southern Biotech, Birmingham, AL, USA). Finally, membranes were rewashed (3 × 15 min), incubated with WEST-ZOL^® chemiluminescence reagent (iNtRON Biotechnology, Seoul, Korea) for 1 min and exposed to film (Blue XB-1, Kodak, Rochester, NY, USA).