Abcc2

The underlying mechanisms of ABCC2 for drug resistance in cell membrane and DCK, TK1, and TK2 for drug metabolism in cytoplasm were evaluated after phytochemicals combination with gemcitabine treatment on T24-GCB cells. Briefly, 10 6 T24-GCB cells after phytochemicals combination with gemcitabine or phytochemicals and gemcitabine alone treatment were trypsinized and washed with PBS twice. Cells were suspended in 100 μL of RIPA buffer (Millipore) contained with cocktail protease inhibitor (Roche, Switzerland). 50 μg protein was electrophoresed for 2 hours in 10% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (PVDF, Millipore) by electro blotter with 100 voltages for 1 hour at 4 C. Antibodies ABCC2 (Cell signaling Technology), DCK (Gene Tex Inc), TK1 (Gene Tex Inc), TK2 (Abcam, UK) were diluted in TBST containing 5% nonfat milk and membranes were incubated for 1 hour with gentle agitation.
The blots were washed for three times with TBST and incubated with goat anti-rabbit antibody conjugated to horseradish peroxidase (Cell signaling Technology) for 1 hour. After three successive washes with TBST (tris-buffered saline (TBS) and Tween20), western blotting chemiluminescence reagent (Bio-Rad, California) was used for protein detection. The membrane was pictured and analyzed by UVP (LLC).