Cdc2 p34

Honokiol (98% purity) was purchased from Nacalai Tesque (Kyoto, Japan). Thiazolyl blue tetrazolium bromide (MTT) and other chemicals of analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO). Leupeptin, pepstatin, and cell proliferation ELISA kit were purchased from Roche Diagnostics GmbH (Mannheim, Germany). Primary antibodies for procaspase 3, caspase 8, caspase 9, CDK6, p53, and cleaved PARP were purchased from Cell Signaling Technology (Beverly, MA). Primary antibodies for cyclin D1, cyclin D2, cyclin E, CDK-2, CDK-4, cyclin A, Cdc2p34, PCNA, caspase 3, anti-mouse IgG horseradish peroxidase-linked and anti-rabbit IgG horseradish peroxidase-linked secondary antibodies, and nitrocellulose membranes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Kip1/p27 antibody was purchased from BD-Pharmingen (San Diego, CA), cyclin B1 and anti-Cip1/p21 antibody from Upstate Biotechnology (Lake Placid, NY).

Protein isolation and Western blots were performed using standard protocols as described elsewhere^{41 (link)}. The following primary antibodies were used: R2 (Santa Cruz Biotechnology, Dallas, USA, cat. #: sc-10844, 1:1000), Wee1 (Santa Cruz Biotechnology, cat. #: sc-9037, 1:1000), Anti-Histone H3.3 G34W (RevMAb Biosciences, South San Francisco, USA, cat. #: 31-1145-00, 1:1000), Histone H3K36me3 antibody (Active Motif, Cambridge, UK, cat. #: 61021, 1:1000), Cdc2 p34 (Santa Cruz Biotechnology, cat. #: sc-54, 1:500), Phospho-cdc2 Tyrosine 15 (Cell Signaling, Denver, USA, cat. #: #9111, 1:500), and Cyclin B1 (Santa Cruz Biotechnology, cat. #: sc-245, 1:200). As secondary antibody we used: Anti-Rabbit IgG (Sigma-Aldrich, cat. #: A-9169,1:10000), rabbit anti-goat IgG-HRP (Santa Cruz Biotechnology, cat. #: sc-2768, 1:10000) and goat anti-mouse IgG-HRP (Santa Cruz, cat. #: sc-2005, 1:5000). Anti-β-Actin antibody (Sigma-Aldrich, cat. #: A2228, 1:100000) or Anti-β-Tubulin (Sigma-Aldrich, cat. #: T4026,1:2000) were used to detect β-actin or β-tubulin as a housekeeping protein.

Total protein (50 μg) was electrophoresed on a polyacrylamide gel with SDS and transferred onto a nitrocellulose membrane. Treated and untreated cells were incubated with anti-cyclin B1 1:200, cdc2 p34 1:200, and anti-MSH2 1:250 (Santa-Cruz Biotechnology, CA, USA) and Vinculin 1:10,000 (Sigma-Aldrich, St. Louis, MO, USA). Band analysis was performed with the Image J (Image Processing and Analysis in Java) software program.