Aurkb

Whole cell protein extract was obtained by collecting cells and lysing using RIPA buffer [50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS] with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). 20 ug of protein was loaded into 7–15% SDS-PAGE gels depending on the molecular weight of the desired protein and then transferred onto nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA). Membranes were blocked for 1 hour in 5% milk in Tris buffered saline Tween-20 (TBST) before overnight incubation at 4°C with primary antibody. The antibodies used include N-cadherin (Abcam, Cambridge, MA, ab12221, 1:300), Fibronectin (Sigma-Aldrich, St. Louis, MO, F3648, 1:1000), p53 (Santa Cruz Biotechnology, Dallas, TX, FL-393, 1:1000), FOXM1 (Santa Cruz Biotechnology, Dallas, TX, SC500, 1:200), PAX8 (Proteintech, Rosemont, IL, 10336-1-AP, 1:500). Antibodies from Cell Signaling (Beverly, MA) include AURKB (3094, 1:1000), PLK (4535, 1:1000), BCL2 (2876, 1:1000), and BIRC5a (2808, 1:500).

Cell lysates were extracted with cell lysis buffer (Beyotime Biotechnology, Nantong, China), and protein concentration was measured by BCA Protein Assay Kit (Tiangen CO., LTD, China). At least 20 μg of each protein sample was loaded for immunoblotting and tested by antibodies that against p27, p21, Skp2, c-PARP, c-caspase 3, c-caspase 9, cytochrome c, AURKA, AURKB (Cell Signaling Inc., Danvers, MA, USA), c-Bax, calpain 4 (Santa Cruz Biotechnology, Santa Cruz, USA), AIF (Epitomics, Hanghzou, China), HA-tag, Flag-tag, Actin (Cw Biotech, Shanghai, China).

Protein lysates and western blots were prepared according to routine protocols. Primary antibodies used were: cyclin D1 1:200 (Merck Millipore, cc-12), cyclin B1 1:500 (Neomarker, Thermo Scientific, MS-868), AURKB 1:1,000 (Cell Signaling Technology, 3094), P21 (CDKN1A) 1:500 (BD Biosciences, 556431), TET2 1:1,000 (Abcam, ab124297), tubulin 1:2,500 (Abcam, ab6160), actin 1:2,000 (Santa Cruz, sc7210), and HSP90 1:5,000 (BD Biosciences, 610418). For all western blot data, n specifies biological replicates.

Cells were treated with described reagents for the indicated times, then harvested and suspended in lysis buffer (50 mM HEPES, pH 7.5, 150 nM NaCl, 10 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM Na₃VO₄, 0.1 mM Na₂MoO₄) with protease inhibitor (Complete Mini, Roche Molecular Biochemicals, Mannheim, Germany) by vortexing. Lysates were incubated on ice for 15 minutes, then centrifuged at 6000rpm for three minutes in a microfuge. Supernatant was collected and stored at -80°C until western blotting. Protein concentrations were determined using the Pierce 660nm Protein Assay (Thermo Fisher Scientific, Rockford, IL). Proteins were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using the XCell SureLock^™ Mini-Cell Electrophoresis System with Novex^® protein gels. The resolved proteins were transferred to nitrocellulose membranes using the iBlot^® dry blotting system (Invitrogen, Carlsbad, CA). Proteins were detected by immunoblot with the following antibodies purchased from Cell Signaling Technology: (phospho-HH3 cat# 9701, Histone H3 cat# 9715, p21 cat# 2947, PARP cat# 9542, phospho-AURKB cat# 3094, AURKB cat# 2914, and α-tubulin cat# 2144). MERTK (cat# ab52968) and TYRO-3 antibodies (cat# ab37841) were purchased from Abcam, and the AXL antibody (cat# AF154) was purchased from R&D.