Evidence of caspase-3-like activity in exosomes secreted by reticulocytes, K562, and Daudi cells. (A) Left panel: Reticulocyte exosomes were solubilized, and various protein amounts were assayed for caspase-3-like activity (1-hour incubation) with the use of Z-DEVD-rhodamine 110. Data shown (means of duplicate experiments) represent total DEVDase activities (□) or activities measured in the presence (▨) of the inhibitor (Ac-DEVD-aldehyde [Ac-DEVD-CHO]). Right panel: Similar protein amounts (250 μg) of exosome from K562 cells and Daudi cells were assayed for caspase-3-like activity (1-hour incubation) as described. (B) Caspase-3 was immunoprecipitated by means of rabbit anti-caspase-3 from exosomes of the 3 cell types: Daudi (3 mg protein), K562 (3 mg protein), and reticulocytes (0.6 mg protein). Controls were carried out with the use of Jurkat cells incubated or not for 7 hours with 5 μM actinomycin D to activate caspase-3. The p20 subunit was revealed by Western blot with the use of the same anti-caspase-3 antibody. (C) Daudi cells were cultured in the presence of Z-DEVD-FMK (20 μM) or DMSO (0.2%), as described in “Materials and methods.” Exosomes were collected from the medium and analyzed for the presence of Lyn by Western blot. Cell lysate and exosomes from Daudi cells were also loaded on the gel as migration controls.