Anti aurka

Purified dephosphorylated AURKA_123–403C290,393A stored at −80°C (25 μM stock solution) was defrosted at RT and diluted to the appropriate concentrations in the kinase assay buffer [20 mM tris-HCl (pH 7.5), 100 mM NaCl, 20 mM MgCl₂, 1 mM DTT, and BSA (0.1 mg ml⁻¹)]. The diluted proteins were then mixed with appropriate concentrations of TPX2_1–43, CEP192_506–536, or CEP192_1–995 (stock concentration: 25 μM) to a total volume of 9 μl. The kinase reaction was initiated by adding ATP (Promega; from 10 mM stock solution to a final concentration of 1 mM) to a total volume of 10 μl. After incubation at RT for 10 min, reactions were stopped by adding 2% SDS. The reactants were run on a 12% SDS–polyacrylamide gel electrophoresis gel and electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked for 30 min at RT with blocking buffer (tris-buffered saline containing 0.1% Tween 20 and 5% skim milk). The membranes were then probed with the indicated primary antibodies (anti–phospho-Thr²⁸⁸ AURKA, Cell Signaling Technology, #3079, 1:20,000; anti-AURKA, Invitrogen, #MA4-58900-A647, 1:10,000) and horseradish peroxidase–conjugated secondary antibodies (goat anti-rabbit, Abcam, #ab97051, 1:10,000; goat anti-mouse, Santa Cruz Biotechnology, #sc-5612, 1:10,000).