Effect of SphK1 expression on signaling pathways. RBL-2H3 cells stably expressing vector or SphK1 were cultured in the absence (A,C) or presence of 15% serum (B,D) for 16 hours. Cells were then treated with S1P (100 nM) for 2 or 10 minutes. Thirty micrograms (A,C) or 60 μg (B,D) of cell-lysate proteins were analyzed by Western blotting with phospho-ERK1/2 (A,B) and phospho-p38 (C,D) antibodies. Blots were stripped and reprobed with antitubulin to show equal loading. Similar results were obtained in 2 additional experiments. (E) SphK1 overexpression attenuates calcium mobilization in response to S1P. RBL-2H3 cells stably expressing vector (□) or SphK1 (▪) were cultured in the presence of serum and loaded with the calcium indicator Fura red-AM for 1 hour at 37°C as described in “Calcium measurements.” Changes in [Ca²⁺]_i after treatment with either S1P (100 nM) or IgE/Ag (100 ng/mL) were measured spectrofluorometrically. Data are expressed as means ± SD of triplicate determinations. Similar results were obtained in 2 additional experiments. ^*P < .01; Student t test. (F) Effect of SphK1 expression on S1P receptor levels. RBL-2H3 cells stably expressing vector or SphK1 were cultured in the absence or presence of 15% serum for 24 hours. Equal amounts of cell-lysate proteins were examined by immunoblotting with anti-S1P₁ or anti-S1P₂ antibodies. Blots were stripped and reprobed with antitubulin antibody to show equal loading.