Fig. 2. Lack of JNK and p38 activation but not apoptotic alteration in ASK1–/– cells in response to anti-Fas (Jo2) treatment. (A) JNK and p38 activation in thymocytes. Cells were isolated from 6-week-old mice, preincubated in serum-free RPMI medium for 4 h and treated with 5 µg/ml Jo2 for the indicated period. Cell lysates were subjected to western blot analysis with antibodies specifically recognizing the activated forms of JNK (phospho-JNK) or p38 (phospho-p38). Membranes were reprobed with antibodies to total JNK or p38 as loading controls. (B) Graphic representation of (A). Fold activation was calculated by dividing the intensity of phosphorylated p55 JNK or p38 at different time points by that at time zero. Essentially similar results were obtained in three independent experiments. (C) Fluorescence analysis of apoptosis of Jo2-treated thymocytes from ASK1+/+ (left panels) or ASK1–/– mice (right panels). Cells were isolated from 6-week-old mice, suspended in serum-free RPMI medium, and cultured in the presence (lower panels) or absence (upper panels) of 5 µg/ml Jo2 for 8 h. Cells were analyzed by FACS after staining with Annexin V and propidium iodide (PI). Percentages of positive cells within a quadrant are indicated. Data are from one of more than five representative experiments.