Anti aurora b

Immunoblotting and immunoprecipitation were performed as previously described^{52 (link)}. Anti-MSK1, anti-Aurora B, anti-β-actin, anti-NFATc2, anti-GADPH, anti-α-tubulin, anti-laminA/C were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-H3S10 and anti-H3 were from Active Motif (Carlsbad, CA, USA). Anti-STAT3, anti-AKT, anti-p42/44, anti-p-STAT3 Y705, anti-p-STAT3 S727, anti-p-AKT S483, anti-p-p42/44 were from Cell Signaling Technology (Danvers, MA, USA).

Glioma cells in the dishes were lysed directly with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN3, 1% Triton X-100, 1 mM PMSF and 1 µg/ml aprotinin, 1 µg/ml leupeptin). The protein concentration was assayed by BCA Protein Assay (Pierce, Rockford, IL). Cell lysates containing equal amounts of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto nitrocellulose (NC) membranes (Millipore Billerica, MA). After overnight blocking with 5% non-fat dry milk in Tris-buffered saline (TBS) at 4°C, then the membranes were incubated with the following primary antibodies: anti-Aurora A, anti-Aurora B, anti-Aurora C (Santa Cruz, CA, USA), anti-Bcl-2, anti-Cyclin D1 and anti-Caspase-3 were purchased from Cell Signaling (Cell Signaling, Danvers, MA) and anti-β-actin (Sigma) at 4˚C overnight and horseradish peroxidase HRP-conjugated secondary antibodies (Jackson ImmunoResearch, PA) for 2 h at room temperature. The protein was visualized with ECL detection reagents (BioVision, CA, USA) and then was examined through scanning densitometry using Tanon Image System. anti-β-actin antibody was used loading control. The experiment was repeated at least three times.