Mutation of the PS binding site in SphK2 attenuates release of SphK2 during apoptosis. (A-C) HEK293 cells were transfected with HA-SphK2 or HA-SphK2-T219A. (A-B) Transfectants were left as controls or treated with Sts. (A) Extracellular SphK2 activity is shown. Data are means ± SEM from 5 independent experiments. ***P < .001. (B) Western analysis showing intracellular expression of HA-SphK2 and the T219A mutant from the same gel. (C) Western analysis showing extracellular t-SphK2 expression in transfectants stimulated with Sts. (B-C) The histograms show quantification of Western data as means ± SEM from 4 independent experiments. (D-E) HEK293 cells, seeded on cover slips, were transfected with (D) HA-SphK2 or (E) HA-SphK2-T219A. Cells remained as controls or were treated with Sts for 2 or 12 hours. Cells were fixed and stained for HA (green) and caspase-1 (red). Nuclear staining was performed with DAPI (blue). White arrows indicate nuclear fragmentation. Each experiment was performed 4 times, and representative images are shown.