Mitochondrial AIF is released into the cytosol before cytochrome c in activated T cells subjected to CD2 apoptotic induction.
(A) Cytosolic and mitochondrial extracts (5 μg) were prepared from cell populations that have been fractionated on Percoll density gradients after a 2-hour apoptotic induction with anti-CD2 or with anti-CD95. F3 and F4 cells were pooled to gain sufficient material. Total cell lysates were obtained from control activated F2-I cells. Immunoblots were probed with anticytochrome c (Cyt.c), with anticytochrome c oxidase subunit II (Cyt.c.ox.II) to detect the presence of mitochondrial material, or with antiactin to monitor protein loading in cytosolic extracts. (B) Specificity of AIF detection by immunoblot analysis. Total lysates of 5 × 105activated T cells (F2-I) were probed with a preimmune rabbit serum (lane 1), or with a rabbit AIF antiserum (lane 2), or with the same AIF antiserum preincubated for 2 hours at 4°C under agitation with 1 mmol/L of a mixture of immunogenic AIF-derived peptides (amino acids 151-170, 166-185, 181-200). (C): Immunoblot analysis of cytosolic extracts from anti-CD2 and anti-CD95 treated cells by using AIF antiserum. Recombinant AIF (5 ng) and total cell lysates (5 μg) were used as positive controls. Data are representative of 5 experiments.
