Anti mdr1

Cytoplasmic cell fraction was collected by using cell lysis buffer (20 mM Tris-HCl (pH 8.0; Wako), 2 mM EDTA (Wako), 0.5% NP-40 (Wako), 1 μM pepstatin (Sigma), 1 μM leupeptin (Sigma), 2 mM sodium orthovanadate (Wako), 1 μM calpain inhibitor (Sigma), phosphatase inhibitor cocktail I/II (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma)). The protein contained amount of these fraction was evaluated using a bicinchoninic acid protein-assay kit (Wako). The extracts (40 μg of protein) were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinyl difluoride membranes (GE Healthcare, Buckinghamshire, UK). The membranes were reacted with the following antibodies: anti-Bcl-2, anti-Bcl-xL, anti-Survivin, anti-MDR1, anti-BCRP, anti-MRP1, anti-LRP1 (Santa Cruz Biotechnologies, CA, USA), anti-phospho-Src (Tyr527), anti-Src (Cell Signaling Technology, Beverly, MA), and anti-β-actin (Sigma) as an internal control. The membranes were reacted with horseradish peroxidase-coupled secondary antibodies (GE Healthcare) for 1 h at room temperature and proteins were assessed using a Luminata Forte (Merck Millipore, Nottingham, UK).

Ice‐cold NP‐40 buffer (100 mmol/L Tris, pH 7.4, 80 mmol/L NaCl, 10 mmol/L EDTA, 0.5% Nonidet P‐40, 0.1% SDS) with proteinase inhibitor mixture (Sigma‐Aldrich) was used to extract the protein. The concentration of each protein samples was measured by DC protein assay kit (Bio‐Rad Laboratories, Hercules, CA). 50 ug of protein for each sample was resolved on SDS–PAGE and transferred to nitrocellulose membrane (Invitrogen). The membrane was next incubated overnight at 4°C with primary antibody, followed by incubation with a corresponding horseradish peroxidase‐conjugated secondary antibody (diluted 1:5000; Santa Cruz Biotechnology). Membrane was developed in West Pico SuperSignal chemiluminescent substrate (Pierce). Primary antibodies used were as follows: anti‐MRP (diluted 1:1000, sc‐130065, Santa Cruz Biotechnology, Inc.), anti‐MDR1 (diluted 1:1000, sc‐55510, Santa Cruz Biotechnology, Inc.), antibeta actin (diluted 1:3000, sc‐28287, Santa Cruz Biotechnology, Inc.), anti‐LRP (diluted 1:1000, sc‐23917, Santa Cruz Biotechnology, Inc.), anti‐Trps1 (diluted 1:1000, SC‐26976, Santa Cruz Biotechnology), and anti‐MGMT (diluted 1:1000, #2739, Cell Signaling Technology, Inc.).