FIGURE 3.

Phosphorylation of Sph and FTY720 in SPHK1- or SPHK2-expressing cells. A, chemical structures of S1P and FTY720-P are indicated. B, expression of SPHK1 and SPHK2. Cell lysates prepared from CHO/mock (mock), CHO/SPHK1 (SPHK1::HA), or CHO/SPHK2 (SPHK2::HA) cells were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Western blot analysis was performed using anti-HA and anti-SPHK1 antibodies. Both SPHK1 and SPHK2 stably expressed in CHO cells were tagged at the C terminus with the HA epitope. *, bands not derived from the tagged SPHK1/2 proteins. C, S1P and FTY720-P synthesis in SPHK1- or SPHK2-overexpressing cells. CHO (mock), CHO/SPHK1 (SPHK1), or CHO/SPHK2 (SPHK2) cells were incubated with the releasing medium containing 5 μm Sph or FTY720 for 2 h. The cells were collected, and the S1P and FTY720-P contents were measured by HPLC. C₁₇-S1P was used as the internal standard. Experiments were performed more than three times, and error bars indicate the S.D.