Figure 5.

Stress-induced nSMase1 Ser-270 phosphorylation, nSMase activation, and ceramide generation in human Jurkat T cells. (a) The phosphorylation of Ser-270 nSMase1 was detected in Jurkat T cells. Cells were heat-shocked at 43 °C for 0, 15, 30, or 60 min and were then recovered at 37 °C for up to 2 h. For UV irradiation, the cells were irradiated by 254 nm UV at 5 mJ/cm². For H₂O₂ treatments, cells were incubated in the presence of 1 mM H₂O₂. For Fas stimulation, cells were treated with 50 ng/ml of anti-Fas antibody and incubated at 37 °C for 0, 15, 30, 60, 120, or 180 min. The cell lysates were analyzed by western blotting with anti-phospho-Ser-270 nSMase1, anti-nSMase1, anti-phospho-MKK7, anti-phospho-MKK4, anti-phospho-JNK, anti-JNK, anti-phospho-c-Jun, or anti-actin antibodies, as indicated. Molecular weight markers are shown in kDa on the right. Asterisks indicate nonspecific signals. (b) The effect of heat shock, ultraviolet light (UV), hydrogen peroxide (H₂O₂), and anti-Fas antibody (Fas) on nSMase activity in Jurkat T cells. (c) The effect of heat shock, UV, H₂O₂, and Fas on ceramide generation in Jurkat T cells. Values represent the means of three independent experiments, and the error bars represent the S.D.s. *P<0.01 versus the control