Down-regulation of SphK1 decreases antigen-induced degranulation and cytokine and chemokine secretion from human LAD2 mast cells. LAD2 cells were transfected with control siRNA (□) or SphK1 siRNA (■) as described in “siRNA transfection.” (A) Equal amounts of lysates (10 μg) were immunoblotted with anti-SphK1 or anti-SphK2 antibodies. Blots were stripped and reprobed with antitubulin to ensure equal loading and transfer. RNA was isolated and mRNA levels of SphK1 were determined by quantitative real-time PCR. Data are expressed relative to cells treated with control siRNA, calculated according to the 2^−ΔΔCt method. (B-F) Duplicate cultures were treated with vehicle, IgE/Ag (30 ng/mL), S1P (100 nM), or ionomycin (1 μM) for 2 hours (B-D,F) or 30 minutes (E). Degranulation (B) and secretion of TNF-α (C), CCL2 (D,E), and IL-6 (F) was determined by ELISA. Data are the means plus or minus SD of triplicate determinations. Similar results were obtained in 3 independent experiments. *P ≤ .01.