Expression of SphK1 increases intracellular S1P and its secretion. (A-C) RBL-2H3 cells stably expressing vector or SphK1-GFP were cultured for 24 hours in the absence (□) or presence of 15% serum (▦) and SphK1 activity was measured (Ai). (Aii) Equal amounts of cell-lysate proteins were also separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-GFP antibody. Blots were stripped and reprobed with antitubulin to show equal loading. Ratios of SphK1/tubulin intensities in the absence and presence of serum are 0.85 and 0.87, respectively, determined with NIH ImageJ.21 Mass levels of cellular (B) and secreted S1P (C) were measured in duplicate cultures as described in “Measurement of S1P.” ^*P < .05 by Student t test. (D-E) SphK1 is not secreted by RBL-2H3 cells. (D) RBL-2H3 cells stably expressing vector or SphK1 were cultured in the absence (-) or presence (+) of 15% serum for 24 hours and (D) SphK1 activity was determined in cells and media as described in “Sphingosine kinase assay.” Data are means ± SD of triplicate determinations. (E) Equal amounts of proteins were analyzed by immunoblotting with anti-GFP antibody. Blots were stripped and then reprobed with antiactin antibody as a loading control. Note the presence of actin in both vector and SphK1 supernatants. The double-headed arrow indicates SphK1-GFP. (F) Longer exposure of a portion of the immunoblot shown in panel E.