*4.8. Immunoblotting*

Expression of ABCG2 protein in whole-cell lysates and plasma membrane vesicles was assessed using immunoblotting as described previously [36], with minor modifications. In brief, the prepared samples were mixed with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer solution containing 10% 2-mercaptoethanol, separated by electrophoresis on polyacrylamide gels, and then transferred to Polyvinylidene Difluoride membranes (Immobilon; Millipore, Billerica, MA, USA) by electroblotting at 15 V for 60 min. For blocking, the membrane was incubated in Tris-buffered saline containing 0.05% Tween 20 and 3% BSA (Nacalai Tesque) (TBST-3% BSA). After overnight incubation at room temperature, blots were probed with rabbit anti-EGFP polyclonal antibodies (A11122; Life Technologies; diluted 1,500 fold in TBST-0.1% BSA), a rabbit anti-α-tubulin antibodies (ab15246; Abcam, Cambridge, MA, USA; diluted 1,000 fold), or a rabbit anti-Na+/K<sup>+</sup> - ATPase α antibodies (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1,000 fold) followed by incubation with a donkey anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated antibody (NA934V; diluted 4,000 fold for EGFP-ABCG2 or 3,000 fold for α-tubulin and Na+/K<sup>+</sup> -ATPase). HRP-dependent luminescence was developed using ECLTM Prime Western Blotting Detection Reagent (GE Healthcare UK, Buckinghamshire, UK) and detected using a multi-imaging Fusion Solo 4TM analyzer system (Vilber Lourmat, Eberhardzell, Germany).
