Anti cyclin b1 h 433

Normal rabbit IgG and anti-actin (I-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal anti-human vinculin was purchased from Sigma-Aldrich (Oakville, ON, USA). Rabbit anti-phospho-Tyr15 CDK1, rabbit anti-phospho-Ser345 CHK1, Bcl-xL and anti-caspase-3 antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-CHK1 (D-7) and anti-cyclin B1 (H 433) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal p53 antibody (CM5) was from Leica Microsystems (Concord, ON, USA), anti-acetyl-p53 (Lys 373, Lys 382) was from Millipore (Billerica, MA, USA) and anti-COX IV was from Abcam (Toronto, ON, USA). Etoposide, PFT-α and PES were purchased from Calbiochem (La Jolla, CA, USA). Fetal bovine serum and Protein-G Agarose beads were purchased from Invitrogen (Carlsbad, CA, USA).

Cultured cells were washed twice with 1X PBS and then incubated for 1 min in urea buffer (8 M urea, 100 mM NaH₂PO₄, and 10 mM Tris pH 8), scraped, harvested and briefly sonicated (10 s). The proteins were subjected to SDS–polyacrylamide gel electrophoresis. The resolved proteins were blotted overnight onto nitrocellulose membranes, which were then blocked in 1X PBS containing 5% non-fat milk for at least 1 h. The blots were incubated with the following primary anti-human antibodies: rabbit polyclonal anti-Cyclin B1 (H433; Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-P53 (DO-7; Dako, Glostrup, Hovedstaden, Denmark); mouse monoclonal anti-Transferrin (clone #507506; R&D Systems, Minneapolis, MN, USA); mouse monoclonal anti-HMOX1 (sc-136960; Santa Cruz Biotechnology, Santa Cruz, TX, USA); mouse monoclonal anti-GAPDH (6C5; Millipore, Burlington, MA, USA); and mouse monoclonal anti-HSP 72/73 (Ab1-W27; Calbiochem, San Diego, CA, USA). The membranes were then incubated for 45 min with the appropriate secondary antibody: donkey anti-rabbit IRdye800 (LI-COR Biotechnology, Lincoln, NE, USA) or donkey anti-mouse IRdye800 (LI-COR). The membranes were then analysed with a LI-COR Odyssey Infrared Image System in the 800 nm channel.