Down-regulation of SphK2 does not alter antigen-induced degranulation, but impairs antigen-induced cytokine secretion from human LAD2 mast cells. LAD2 cells were transfected for 48 hours with control siRNA (□) or SphK2 siRNA (). (A) Equal amounts of lysates (10 μg) were immunoblotted with anti-SphK2. Lysate from cells overexpressing SphK2 was loaded as a positive control (left lane). Note that due to the high level of expression of SphK2, this lane from the same gel was developed for a much shorter time; a vertical line has been inserted to indicate this distinction. Blots were stripped and reprobed with antitubulin to ensure equal loading and transfer. RNA was isolated and SphK2 mRNA levels were determined by quantitative real-time PCR. Data shown are relative to cells treated with control siRNA, calculated according to the 2^−ΔΔCt method. (B-E) Duplicate cultures were stimulated with vehicle, IgE/Ag (30 ng/mL), S1P (100 nM) or ionomycin (1 μM). Degranulation (B) and secretion of TNF-α (C) and IL-6 (D) were determined after 2 hours, and secretion of CCL2 (E) was determined after 30 minutes. Data are the means plus or minus SD of triplicate determinations. Similar results were obtained in 3 independent experiments. *P ≤ .01.