Monoclonal antibodies against gapdh

MRC‐5 cells were lysed, the proteins were extracted, and Western blotting was performed (Towbin & Staehlin, 1979; Towbin, Staehelin, & Gordon, 1979). The immunoblots were washed with Western blot stripping buffer (pH 2–3; Nacalai, Tokyo, Japan) and probed with monoclonal antibodies against GAPDH (1:2,000; Proteintech Group, Chicago, IL, USA).

Protein assays were performed according to the Bradford method using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8 % acrylamide gels, and then transferred to Hybond™ membranes (Amersham, Germany). The membranes were blocked overnight in 5 % skimmed milk in Tris-buffered saline with Tween®-20 (TBST). For immunoblotting, the membranes were incubated at 4 °C overnight with anti-MDR1 (Bioss, Peking, China) and anti-GST-π, anti-MRP1 (Proteintech Group, Chicago, USA) antibodies, rinsed with TBST, and incubated with anti-rabbit IgG antibodies conjugated to horseradish peroxidase (HRP; Dako, Carpinteria, CA, USA) at a dilution of 1:5000. After applying electrochemiluminescent (ECL)-Plus detection reagents (Santa Cruz, CA, USA), the protein bands were visualized using an X-ray film (Fujifilm, Tokyo, Japan). The immunoblots were washed with Western blotting stripping buffer (pH 2–3; Nacalai, Tokyo, Japan) and probed with monoclonal antibodies against GAPDH (1:2000; Proteintech Group, Chicago, USA).