ASK1 activation is involved in ceramide-induced apoptosis. (A) Jurkat T cells were treated with 20 μM C2-ceramide for different time periods as indicated. The expression of phospho-ASK1 at serine 83 (S83 pASK1) and at threonine 845 (T845 pASK1), and ASK1, were determined using Western blot analysis. The relative ratio of pASK1 to total ASK1 is shown. The expression of α-tubulin was an internal control. (B) The cell lysate from untreated or 20 μM C2-ceramide–treated Jurkat T cells for different time periods was immunoprecipitated using antithioredoxin antibody, followed by immunoblotting using anti-ASK1 and antithioredoxin antibodies. Cell lysate without ceramide treatment and immunoprecipitation (WCL indicates whole-cell lysate) was used as a positive control; mouse IgG (first lane) was the negative control. (C) Vector control– and hTxnip siRNA-transfected Jurkat T cells were treated with 20 μM C2-ceramide for 6 hours, and the expression of T845 pASK1 was measured by flow cytometric analysis. The percentages of positive cells are shown as means plus or minus SD of triplicate cultures (***P < .001 as compared with the vector control group). (D) ASK1 protein expression in vector control– and ASK1 siRNA-transfected Jurkat T cells were detected by Western blotting (left). The relative ratio of ASK1 to EGFP is shown. The expression of α-tubulin was used as an internal control. Vector control– and ASK1 siRNA-transfected cells were treated with 20 μM C2-ceramide for 24 hours, and cell apoptosis was measured by annexin V staining. The percentages of apoptotic cells are shown (means ± SD of triplicate cultures; ***P < .001 as compared with the vector control group).
