Kinetics of cytochrome c release during ATG-induced apoptosis of T cells. PBLs were incubated with NRS, rabbit ATG (250 μg/mL), or horse ATG (250 μg/mL). (A) At the indicated times, cytosolic extracts were prepared and proteins were separated on 15% SDS-PAGE followed by Western blotting with the anti-cytochrome c mAb or the anti-cytochrome oxidase mAb. Anti-cytochrome oxidase served as a marker of mitochondrial contamination of the extracts. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti-β-actin mAb. Apoptosis (%) was measured by surface binding of annexin V at the indicated times. (B) After 24-hour treatment, cytochrome c release was visualized by immunofluorescence staining. Results are from one representative experiment of 2 independent experiments showing similar results. Original magnification, × 63.