Western blot analysis of Yama/CPP32/Apopain in FVA cells and Hu-ECFCs. (A) The lettering at the top of the panels shows the time of culture of FVA cells (2, 4, 8, or 12 hours) without EP (−) or with EP (+). All cell samples were prepared as described and subjected to electrophoresis on a 15% acrylamide gel. The upper panel shows depletion of the 32-kD proenzyme over 12 hours without EP (30 seconds of film exposure). The lower panel shows accumulation of the 17-kD apopain large peptide in cells cultured without EP (4 minutes of film exposure). (B) The panel shows the cleavage of the 32-kD proenzyme that occurs upon EP deprivation of Hu-ECFCs. Day-8 Hu-ECFCs were cultured for 24 hours without or with EP (day-9 EP− or day-9 EP+). The results of two different preparations of cells are shown. The 32-kD and 17-kD bands are labeled. All blots were probed using 1 μL of the rabbit polyclonal antiapopain antibody.