Anti ube2c

Frozen patient tissues were homogenized in 1X RIPA buffer (Millipore, Billerica, MA, USA) supplemented with 1X Complete protease inhibitor cocktails (Roche, Mannheim, Germany). The homogenates were briefly sonicated and centrifuged at 14000rpm at 4°C for 15 minutes. The supernatant (tissue lysate) was transferred to a new vial, and 30 μg of tissue lysate from each sample was resolved by 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in 1X TBST and then probed with primary antibodies at dilutions suggested by the manufacturers. Anti-DSN1, anti-SKA3, anti-UBE2C, anti-cyclin D, and anti-cyclin B1 antibodies were purchased from Abcam (Cambridge, MA, USA), anti-Aurora A was from Cell Signaling Technology (Danvers, MA, USA), anti-cyclin A and anti-cyclin E were from Santa Cruz (Santa Cruz, CA, USA), and anti-GAPDH was from Proteintech (Chicago, IL, USA). Immunoblotting images were quantified using GeneTools software (SynGene Inc., Frederick, MD, USA), and intensities of the target proteins were normalized to GAPDH.