Anti α tubulin sc 8035

Anti-Pin1 (sc-46660, RRID: AB_628132), anti-α tubulin (sc-8035, RRID: AB_628408) and anti-actin (sc-8432, RRID: AB_626632) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-active caspase-3 (9661, RRID: AB_2341188) antibody was purchased from Cell Signaling (Boston, MA, 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).