Anti cks1

Western blot analysis was performed as described elsewhere.^{11 (link)} Briefly, the siRNA-transfected cells and negative control cells were washed with ice-cold phosphate-buffered saline (PBS) and homogenized in cell lysis buffer. After centrifugation for 10 min at 4 °C, the supernatant was harvested and the protein concentration was measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Supernatant proteins (30 μg per lane) were electrophoresed in 10% SDS-polyacrylamide gel and were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), then incubated with the following primary antibodies: anti-KIF14 (1:1000, Abcam, Cambridge, UK), anti-p16, anti-p21, anti-cyclin D1, anti-cyclin B1, anti-cyclin E1 (1:1000, Epitomics, Burlingame, CA, USA), anti-p27 (1:1000, BD Biosciences, San Jose, CA, USA), anti-Skp2 (1:1000, BD Biosciences), anti-Cks1 and anti-α-tubulin (1:1000 to 1:3000, Santa Cruz Biotechnology, Dallas, TX, USA). The membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h and visualized using the ECL detection kit (Amersham-Pharmacia Biotech, Braunschweig, Germany). Alpha-tubulin and beta-actin were used as internal controls for western blot analysis.