Anti ube2c

Cell protein extraction was performed by washing them twice in ice-cold PBS and subsequently lysing them by using RIPA-like buffer (250 mM NaCl, 50 mM TRIS-HCl, pH 7.4, 0.1% SDS, 2 mM DTT and 0.5% NP-40, (Sigma, St. Louis, MO, USA)) containing protease inhibitors (Roche, Belmonte, CA, USA). Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA) and using bovine serum albumin was employed as standard. An amount of 50 µg of total protein extract was resolved onto a 8.0% SDS PAGE, transferred a nitrocellulose-membrane (Roche) and probed with the appropriate antibodies for 1 h. Antibodies anti-FOXM-1 (Santa Cruz), anti-UBE2C (Boston Biochem, Cambridge, MA, USA) and anti-tubulin (Sigma, St. Louis, MO, USA) were used at 1:250, 1:500 and 1:1000 dilutions, respectively. Next, membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000) for 1 h and detection was performed with enhanced chemiluminescence ECL Kit (Amersham, Piscataway, NJ, USA).