Abcb1

Total cell lysate extraction and western blotting were performed using a previously described protocol.14, 15 The extracted protein was quantitated using a protein assay (Bio‐Rad Laboratories) and was transferred to a membrane using a transfer system (Bio‐Rad Laboratories, Hercules, CA, USA). Then, the membranes were probed with primary antibodies diluted with 5% BSA overnight at 4°C. After washing more than 3 times using PBS‐tween, the membranes were incubated with the secondary antibodies for 1 hour at 25°C. To detect specific signals, we examined the membrane using the ECL Prime Western Blotting Detection System (GE Healthcare) and LAS‐3000 (Fuji Film). The primary antibodies were as follows: p‐HER2 (Tyr1221/1222), HER2, p‐EGFR (Tyr1068), EGFR, p‐MAPK (Erk1/2) (Thr202/Tyr204), MAPK (Erk1/2), p‐AKT (Ser473), AKT, IGF‐I receptor (IGF‐1R), p‐IGF‐I receptor (phospho‐IGF‐1R) (Tyr1135/1136), MET, p‐MET(Tyr1234/1235), YES, Src, p‐Src (Tyr416), ALDH1A1, ABCB1, E‐cadherin, vimentin and p‐AXL(Tyr702) (Cell Signaling Technology, Danvers, MA, USA), AXL (R&D Systems, Minneapolis, MN, USA), and actin (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies used in this study were as follows: goat anti–mouse IgG‐HRP, goat anti‐rat IgG‐HRP and donkey anti–goat IgG‐HRP (Santa Cruz Biotechnology).

Western blot analysis was performed on cell extracts of the indicated cell lines that had been transfected for 48 h with the miRNA sequences. Immunoblots were performed from whole cell lysate prepared using RIPA Buffer supplemented with PMSF, and phosphatase inhibitors. Cell lysates were quantified for protein content using a bicinchoninic acid protein assay kit. SDS PAGE was used to separate the proteins, with 30 µg total protein per lane on 5% SDS-PAGE. Protein samples were transferred to 0.22-mm polyvinyl difluoride membranes. After saturation in Tris-buffered saline with Tween-20 and 5% powdered milk, the membranes were incubated with anti-eIF4G (cat. no., 2617S; dilution, 1:1,000) and anti-β-actin (cat. no., 8457S; dilution, 1:1,000) antibodies purchased from Cell Signaling Technology, ABCB1 (cat. no., ab170904; dilution, 1:1,000), ABCC1 (cat. no., ab3368; dilution, 1:1,000) and ABCG2 (cat. no., ab108312; dilution, 1:1,000) purchased from Abcam, overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no., sc-2357; dilution, 1:5,000 Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature. The signals were visualized using ECL Substrates (Tanon Ltd, shanghai, China) and quantified using AlphaImager 2200 software.