Transport assays were performed based on the rapid filtration protocol established by Leier et al. 29 (link). ABCC11 and control membrane vesicles, respectively (50 μg; Gibco, Darmstadt, Germany), were prepared in buffer containing 50 mm D-mannitol, 50 mm TRIS-Base, pH 7.0, 8 mm K2CO3, 10 mm MgCl2, 10 mm phosphocreatine, 4 mm ATP or AMP, 75 mU phosphocreatine kinase and radiolabelled substrate in a total volume of 75 μl. The following radiolabelled substrates were used: [estradiol–6,7–3H(N)]-17–β–D–glucuronide, [3H–(G)]taurocholic acid, [14,15,19,20–3H(N)]-leukotriene C4 and [14,15,19,20–3H(N)]-leukotriene D4 (all from Perkin Elmer, Waltham, MA, USA) and [8–3H]cyclic guanosine–3′,5′–monophosphate (Hartmann Analytic, Braunschweig, Germany).
Samples were incubated at 37°C, and 20 μl were removed after 5, 30 and 60 s. The transport was stopped by transferring the samples in 1 ml stop solution (50 mm D-mannitol, 50 mm TRIS-Base, pH 7.0). Using vacuum filtration, samples were sucked through 0.22 μm GVWP filters (Millipore, Billerica, MA, USA). Filters were washed five times with 1 ml stop solution and transferred into scintillation vials. Radioactivity was assessed using a liquid scintillation counter. Net ATP-driven transport was determined by subtracting values of the corresponding control sample, containing AMP, from the transport in the presence of ATP.
Samples were incubated at 37°C, and 20 μl were removed after 5, 30 and 60 s. The transport was stopped by transferring the samples in 1 ml stop solution (50 m