Sc 246

Cells or tissues lysates were separated by SDS-poly-acrylamide gel electrophoresis and transferred to poly-vinylidene difluoride membranes (Amersham). Membranes were blocked with 5% non-fat milk and then probed with antibodies against SOX7 (Santa Cruz, sc-20093, 1∶1000), cyclin D1 (Santa Cruz, sc-246, 1∶1000), c-myc (Santa Cruz, sc-40, 1∶1000) and GAPDH (Santa Cruz, sc-32233, 1∶2000) as indicated. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using the ECL system (Amersham). All the experiments were performed independently three times at least.

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), rhodamine 123, dichlorofluorescein diacetate (DCF-DA) and 4′,6-diamidino-2-phenylindole (DAPI), and the antibodies for β-actin and NFκB were purchased from Sigma (St. Louis, MO, USA). Primary antibodies for cyclin D1 (SC-246), caspase 3 (SC-7148), and lamin B (SC-6217) and secondary antibodies, goat anti-rabbit IgG-HRP (SC-2004), goat anti-mouse IgG-HRP (SC-2005), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells were lysed in lysis buffer (20 mM Tris, pH 7.4, 5 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 1% NP-40, 1 mM PMSF, 0.2% protease inhibitor cocktail and phosphatase inhibitor). The protein concentrations of the cell lysates were measured using the Pierce BCA Protein Assay kit (Pierce, Rockford, USA). Protein lysates were then resuspended in loading buffer, boiled for 5 minutes, subjected to SDS-PAGE, and immunoblotted with antibodies. In addition to CKAP2 antibody, antibodies for phospho-S10-histone H3 (ab14955, Abcam, Cambridge, UK, 1:500), Rb (#9309, Cell Signaling, Danver, MS, 1:2000), phospho-Rb-S807, S811 (#9308, Cell Signaling, 1:1000), cyclin D1 (sc-246, Santa Cruz, Dallas, TX, 1:1000), cyclin E (sc-481, Santa Cruz, 1:1000), cyclin A (sc-751, Santa Cruz, 1:1000), cyclin B1 (#4138, Cell Signaling, 1:1000), and GAPDH (ab8245, Cell Signaling, 1:2500) were used.