*4.10. Urate Transport Assay*

The urate transport assay, with ABCG2-expressing plasma membrane vesicles, was conducted using a rapid filtration technique described in our previous studies [27,36], with some minor modifications. In brief, each plasma membrane vesicle (0.5 mg/mL) was incubated with 20 µM of radiolabeled urate in a reaction mixture (total 20 µL: 10 mM Tris/HCl, 250 mM sucrose, 10 mM MgCl2, 10 mM creatine phosphate, 1 mg/ml creatine phosphokinase, pH 7.4, and 50 mM ATP, or AMP as an ATP substitute) for 10 min at 37 ◦C. After incubation, the reaction mixture was mixed with 980 µL of ice-cold stop buffer (2 mM EDTA, 0.25 M sucrose, 0.1 M NaCl, 10 mM Tris-HCl, at a of pH 7.4); the resulting solution was rapidly filtered through an MF-Millipore Membrane (HAWP02500; 0.45 µm pore size and 25 mm diameter; Millipore). After washing with 5 ml of ice-cold stop buffer five times, the plasma membrane vesicles on the filter were dissolved in Clear-sol II (Nacalai Tesque). Then, the radioactivity in the plasma membrane vesicles was measured using a liquid scintillator (Tri-Carb 3110TR; PerkinElmer, Waltham, MA, USA).

The urate transport activity was calculated as the incorporated clearance (µL/mg protein/min) defined as the incorporated level of urate [disintegrations per minute (DPM)/mg protein/min]/urate level in the incubation mixture [DPM/µL]. ATP-dependent urate transport was calculated by subtracting the urate transport activity in the absence of ATP from that in the presence of ATP; ABCG2-mediated urate transport activity was calculated by subtracting ATP-dependent urate transport activity of control plasma membrane vesicles from that of ABCG2-expressing plasma membrane vesicles.
