Various apoptotic stimuli lead to the extracellular release of cytochrome c.
(A) Jurkat cells (5 × 106) were cultured in growth medium and either left untreated or stimulated for 15 hours with the indicated concentrations of anti-CD95 mAb and staurosporine or for 24 hours with the indicated concentrations etoposide or doxorubicin. Cytochrome c was precipitated from the culture medium (m) and the cellular extracts (c) with antibodies against the native molecule. The immunoprecipitated material was separated by SDS-PAGE and analyzed by immunoblotting. The quantity of cytochrome c was then determined by densitometric analysis. The graphs display the amount of cytochrome c in the culture medium relative to the total content of cytochrome c present in medium and cells. (B) Cytochrome c is released from cells as a soluble protein. Supernatants of Jurkat cells treated for 15 hours with staurosporine were centrifuged for 30 minutes at 10 000g or 100 000g and immunoblotted as described above. Open arrowheads indicate heavy chain and light chain of anticytochrome c antibody; closed arrowhead indicates cytochrome c.
