Activated T-cell death triggered by PSGL-1 cross-linking involves release of mitochondrial mediators. Differential subcellular localization of mitochondrial mediators was analyzed in activated mouse T cells on death triggered by cross-linking of PSGL-1. Immunoblotting with anti-AIF antibody and anti–cytochrome c antibody, respectively, revealed that AIF translocation was first detected in the 3-hour TAB4-treated cytosolic fraction (A). Nuclear fraction of activated T cells at indicated time points following TAB4 treatment was immunoblotted by anti-AIF (B). (C) Cytochrome c release from mitochondria was detected in cytosolic fraction of 6-hour treated cells. The data represent 3 independent experiments.