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. 2000 Mar 20;191(6):1017–1030. doi: 10.1084/jem.191.6.1017

Figure 2.

Figure 2

p38 MAPK and JNK are activated during AICD in DO11.10 T hybridoma cells and primary splenic T cells. (A) DO11.10 T cells were pretreated with the caspase inhibitors YVAD, DEVD, or zVAD and were then cultured in anti-CD3 (10 μg/ml)-coated plates for varying times. The percent of apoptotic cells was determined by PI staining as in Fig. 1. p38 MAPK was immunoprecipitated with an anti-p38 MAPK, and p38 MAPK activity in immunoprecipitates was measured using GST–ATF-2 as substrate. JNK activity was measured using a solid-phase JNK assay. Cell lysates were reacted for 4 h at 4°C with GST–c-Jun precoupled to glutathione–agarose beads, followed by an in vitro kinase reaction. Data are shown as mean values ± SEM and are from one of two independent and reproducible experiments. The relative activities of the p38 MAPK and JNK were quantitated by densitometric scanning and are shown below the respective gel lanes. (B) Splenic T cells from B6 mice were activated with plate-bound anti-CD3 and anti-CD28 mAbs for 48 h. Activated T cells were then restimulated in wells coated with anti-CD3 for 16 h in the presence of the caspase inhibitors YVAD, DEVD, or zVAD. For in vitro kinase assays of p38 MAPK and JNK, activated T cells were restimulated with plated-bound anti-CD3 for 2 h and 8 h, respectively, and the relative activities of p38 MAPK and JNK were assayed and quantitated as in A. (C) Splenic T cells from gld mice (left panel) were activated with plate-bound anti-CD3 and anti-CD28 mAbs for 48 h. Activated T cells were then restimulated in wells coated with anti-CD3 for 2 and 8 h. JNK and p38 MAPK activities were assayed as in A. Cell lysates were immunoblotted with anti–caspase-1 (Casp1) or anti-CPP32 (Casp3), respectively (left panel). Alternatively, B6 splenic T cells (right panel) were activated as in B, and activated T cells were then treated with plate-bound anti-Fas (5 μg/ml) for 8 h in the presence or absence of zVAD (50 μ/M). Activities of JNK and p38 MAPK and cleavage of caspase-1 and -3 were assayed as above. A and B, p29, line 4–5: in the presence of the caspase inhibitors YVAD, DEVD, and zVAD.

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