Figure 5.
Inhibition of cytochrome c release from the mitochondria into the cytoplasm by overexpression of gelsolin. (A) Viability of parental Jurkat cells, a vector-transfected derivative (JNF-2), and derivatives overexpressing gelsolin (JGF-5 and JGF-7) treated with anti-Fas antibody. After plating at a density of 1 × 104 cells per well in 96-well dishes, the cells were treated with an anti-Fas antibody for the indicated periods and viability was assessed morphologically by counting normal round cells as alive and fragmented cells as dead. Three independent experiments were carried out and more than 200 cells were counted in each experiment. (B) Processing of procaspase-3 after Fas-stimulation. Cells were treated with anti-Fas antibody for the indicated periods. Cell lysates (4 × 105 cells per lane) were fractionated by SDS/15% PAGE and immunoblotted with anti-caspase-3 antibodies. (C) Immunoblot analysis of cytochrome c in the membrane and cytosolic fractions. JNF-2 and JGF-5 cells were treated with anti-Fas antibody for the indicated periods, and membrane and cytosolic fractions were prepared as described. Each fraction (equivalent to 4 × 105 cells) was subjected to SDS/15% PAGE and immunoblotting with anti-cytochrome c antibody.