Anti p aurora a thr288

The proteins of whole cell lysates were prepared in RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA), and quantified by the bicinchoninic acid (BCA) method. Immunoblotting analysis of proteins in cell lysates was performed as previously described [26 (link)]. Primary antibodies used were as follows: anti-Aurora A (#12100), anti-p-Aurora A (Thr288) (#3079), anti-Histone H3 (#4499), and anti-p-Histone H3 (Ser10) (#9701), and anti-cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-PARP-1 (#sc-8007) and anti-GAPDH (#sc-25778) were purchased from Santa Cruz (CA, USA).

Cells were cultured on glass slides coated with 0.05 mg/ml PDL (Sigma-aldrich, St. Louis, MO, USA) in the presence of TY-011 (0.8 μM) or equivalent amount of DMSO for 24 h. The cells were fixed in 4% paraformaldehyde at room temperature, then permeabilized with 0.15% Triton-X100 and blocked in 3% BSA in PBS. The primary antibodies were incubated with slides at 1:100 dilution in PBS containing 3% BSA in a wet chamber at 4 °C overnight. The secondary antibodies Alexa Fluor H 488 goat anti-rabbit IgG (H + L) or Alexa Fluor H 594 goat anti-mouse IgG (H + L) (Invitrogen, Carlsbad, CA, USA) were applied respectively for 2 h after the slides were washed with PBS thoroughly at room temperature. After the unbound antibodies were removed, the slides were dried and mounted with DAPI-fluoromount-G (Beyotime, Shanghai, China). The images were captured with FCFM (fluorescence confocal microscope) (Olympus FV1000 IX81, Tokyo, Japan) under appropriate excitation and emission filters or phase-contrast microscope (Olympus IX73, Tokyo, Japan). Primary antibodies used were as follows: anti-p-Aurora A (Thr288) (#3097) and anti-p-Histone H3 (Ser10) (#9701) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-MPM2 (anti-phospho-Ser/Thr-Pro) (#05-368) was purchased from Merck Millipore (Billerica, MA, USA); and anti-α-tubulin (sc-8035) was purchased from Santa Cruz (CA, USA).