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.
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.