FIG. 1.

LPS activates SPK in macrophages. (A) Dose-dependent activation of SPK by LPS. Serum-starved RAW 264.7 cells and HMs were stimulated with different doses of LPS for 40 min. Then, the SPK activity in cell lysate was assayed by measuring [γ-³²P]ATP incorporation into sphingosine-1-phosphate. (For RAW 264.7 cells, relative SPK activity of 100% = 35 pmol/min/mg of protein. For HMs, relative SPK activity of 100% = 32 pmol/min/mg of protein). (B) Time course of SPK activation by LPS. RAW 264.7 cells were serum starved overnight and treated with 1 μg of LPS/ml for the indicated time period. Then, the SPK activity in cell lysate was assayed. (C) Analysis of total cellular sphingosine (Sph) and S1P levels in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were serum starved overnight and treated with 1 μg of LPS/ml for the indicated time period, and then the relative levels of total cellular Sph and S1P were determined.