Rabbit anti mdr1

Protein lysates were prepared by lysing the HCT116/5-Fu cell lines or tumor tissues in lysis buffer [50 mM Tris–HCl pH 6.8, 5 mM EDTA, 2% sodium dodecyl sulfate (SDS), and 5% glycine]. Protein concentration was estimated by BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts (30 µg) of protein samples were fractionated by SDS-polyacrylamide gel electrophoresis on a 12% polyacrylamide SDS gel at 80 V for 2 hours. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 200 mA for 3 hours. The membranes were blocked for 1 hour with 5% skim milk in 0.1% TBST (20 mM Tris pH 7.6, 150 mM NaCl, and 0.05% Tween-20) followed by incubation with primary antibodies at 4°C overnight. The next day, the membranes were washed three times for 15 minutes in TBST, followed by incubation at room temperature with horseradish peroxidase-conjugated antirabbit or antimouse secondary antibody (Santa Cruz Biotechnology Inc., Dallas, CA, USA, dilution 1:5,000). Primary antibodies used rabbit antihuman EIF3G (Bethyl Laboratories, Inc., Montgomery, TX, USA; cat no A301-757A; 1:1,000), mouse antihuman GADPH (Santa Cruz Biotechnology; cat no sc47724; 1:1,000), mouse anti-MRP (Santa Cruz Biotechnology; cat no sc-136447; 1:1,000), and rabbit anti-MDR1 (Abcam, Cambridge, UK; cat no ab129450; 1:1,000).