SphK2 is dispensable for degranulation and migration of hMCs but is required for secretion of TNF-α. Purified cord blood–derived human mast cells (hMCs) were transfected with control siRNA (□) or SphK2 siRNA (). (A) SphK2 mRNA levels normalized to GAPDH mRNA were measured by QPCR. (B-D) hMCs were sensitized overnight with IgE and then treated with Ag (30 ng/mL), S1P (100 nM), or ionomycin (1 μM) for 2 hours. Degranulation (B) and secretion of CCL2 (C) and TNF-α (D) were determined. Data are the means plus or minus SD of triplicate determinations. Similar results were obtained in 3 independent experiments. (E) hMCs were transfected with control siRNA (□), siRNA targeted to SphK1 (■), or siRNA targeted to SphK2 (). Cells were then sensitized with 1 μg/mL anti-DNP IgE for 12 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. *P ≤ .01.