Chemotaxis of mast cells toward antigen and S1P is pertussis toxin sensitive. Migration of (A) unsensitized LAD2 cells toward increasing concentrations of S1P (0.1, 1, 10, 20 μM) or FN (20 μg/mL) and (B) IgE-sensitized LAD2 cells toward increasing concentrations of Ag (10, 30, 100 ng/mL) was determined in transwell chambers. *P ≤ .01 compared with vehicle in unsensitized cells. **P ≤ .01 compared with vehicle in sensitized cells. (C) S1P (10 μM) or Ag (30 ng/mL) was added to either the bottom chamber () or to both the top and bottom chambers (), and migration of LAD2 was determined. (D) LAD2 cells pretreated for 1.5 hours without (□) or with pertussis toxin (PTX, 100 ng/mL, ) were allowed to migrate toward vehicle, S1P (1 μM), IgE/Ag (30 ng/mL), or fibronectin (20 μg/mL). (E) LAD2 cells pretreated for 1.5 hours without (□) or with pertussis toxin (PTX, 100 ng/mL, ) were treated with vehicle, S1P, anti-DNP IgE and DNP-HSA (IgE/Ag), or ionomycin (1 μM), and degranulation was determined by β-hexosaminidase release. (F,G) Chemotaxis of mast cells toward antigen is dependent on SphK1 but not on SphK2. (F) LAD2 cells were transfected with control siRNA (□) or siRNA targeted to SphK1 (■) or (G) siRNA targeted to SphK2 (). Cells were then sensitized with 1 μg/mL anti-DNP IgE for 5 hours, and allowed to migrate toward vehicle, Ag (30 ng/mL), S1P (1 μM), or fibronectin (20 μg/mL) for 24 hours. Data are expressed as percentage migrating cells and are means plus or minus SD. Similar results were obtained in 3 independent experiments.