Western blot

The concentration of total protein extracted from parental and gemcitabine-resistant cells was determined with a BCA Protein Assay Kit (Pierce, USA). Equal amounts of protein were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, Bedford, USA) using a mini trans-blot (Bio-Rad laboratories, Hercules, CA, USA). Rat anti-human CIP2A, p- AKT(Millipore, Bedford, USA), and BCL2 (Abcam, MA, USA) were used to detect the expression of these proteins. β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as an internal control. Electrochemiluminescence was performed according to the manufacturer's instructions and read with a Chemi lmager 5500 imaging system (Alpha Innotech Co, San Leandro, CA, USA).