Aurora kinase b

Cells were harvested, lysed and western blot was performed as previously described^{34 (link)}. Antibodies were used against MELK (#2274), Aurora B kinase (#3094), cyclin B1 (#4138), pFOXM1 (#14170), EZH2 (#4905), Cdc25B (#9525), Plk-1 (#4535), Aurora A kinase (#3092), PARP (#9542), Mcl1 (#5453), pBcl-2 (#2827), Bcl-xL (#2764), caspase-3 (#9662) and β-actin (#4967) (all from Cell Signaling Technology, Leiden, the Netherlands) and FOXM1 (sc_376471), Bcl-2 (sc_492) and pBcl-xL (sc_101644) (Santa Cruz, Heidelberg, Germany). Quantification of the different protein levels was performed in Image J, according to manufacturer’s instructions.

Cells were lysed in lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, and 1 mM Na₃VO₄). Lysates were immunoblotted with the corresponding primary antibodies. Antibodies for hARD1, cyclin B1, cyclin E, p53, and green fluorescent protein (GFP) were purchased from Santa Cruz Biotechnology, and Aurora kinase A (AURKA) and Aurora kinase B (AURKB) were from Cell Signaling.
For the nuclear/cytosolic fractionation, cultured cells were washed with PBS, homogenized in buffer A (10 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.1% NP-40), and centrifuged for 10 min at 600×g in 4°C. For the cytosolic fraction, supernatants were re-centrifuged for 30 min at 15,000 rpm. For nuclear extracts, the nuclear pellet was washed with buffer A, resuspended in buffer C (10 mM HEPES (pH 7.4), 1.5 mM MgCl₂, 0.5 mM DTT, 20% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, and 420 mM NaCl), centrifuged for 30 min at 15,000 rpm, and the supernatant isolated.