Active full-length caspase-1 is needed for SphK2 cleavage during cell death. (A) Apoptosis in HEK293 cells was induced with Sts in the presence/absence of 1μM Ac-YVAD-CMK (C1-I), or with TNF-α. SphK2 activity in the supernatant is displayed. DMS was used as an internal control in the kinase assay. Data are means ± SEM from 4 independent experiments. *P < .05. (B) HEK293 cells were transfected with HA-hSPHK2/pCMV5 (HA-SphK2). Cells were left as controls or treated with Sts alone or in combination with Ac-YVAD-CMK. Histograms show SphK2 activity in the supernatant. DMS was used as an internal control. Data are means ± SEM from 5 independent experiments. ***P < .001. (C-E) HEK293 cells were transfected with HA-SphK2 or different HA-tagged caspase-1 constructs as indicated. Transfectants remained untreated or were incubated with Sts for 6 hours. (C) Western analysis showing expression of HA-SphK2, HA-tagged caspase-1, constructs and caspase-1–p20 in the cytosol as well as SphK2 in supernatants. (D) Western analysis showing expression of HA-SphK2 and HA-tagged caspase-1 constructs by staining for the HA-tag. Vertical lines have been inserted to indicate repositioned lanes from the same gel. The histogram shows quantification of SphK2 expression as means ± SEM from 4 independent experiments. *P < .05. (E) Overexpressed HA-Casp1 and HA-Casp1-C285A were precipitated from crude lysates using an anti-HA antibody. Binding of SphK2 to precipitated HA-tagged caspase-1 was detected by Western analysis. As an input control, crude lysates were analyzed for total amounts of overexpressed HA-tagged caspase-1.