FIGURE 6.

S1P-releasing assay of cells expressing ABC transporters. A, expression of ABC transporters in the cells transfected with each transporter individually. Membrane (m) and soluble fractions (s) prepared from CHO/SPHK1 cells (mock) or CHO/SPHK1 cells stably expressing mABCA1, hABCB1, mABCC1, or hABCG2 were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Western blot analysis was performed with specific antibodies for each individual ABC transporter as described under “Experimental Procedures.” A ubiquitously expressed membrane protein, Na⁺/K⁺-ATPase, was used as the loading control of each membrane fraction. B and C, S1P or FTY720-P secretion from ABC transporter-expressing cells. CHO/SPHK1 cells (mock) or CHO/SPHK1 cells stably expressing mABCA1, hABCB1, mABCC1, hABCG2, or hSPNS2 were incubated with releasing medium containing 5 μm Sph (B) or FTY720 (C) for 2 h. The releasing medium was collected, and the amounts of released phosphorylated sphingolipids were measured by HPLC. Each diagram is a typical chromatogram of the sphingolipids released from the cells overexpressing an individual ABC transporter. C₁₇-S1P was used as the internal standard. Experiments were performed more than three times.