Figure 4. Treatment of mitochondria with bSMase induces Bax association with mitochondria.

Mitochondrial fraction was prepared by fractionation as described in the Experimental section, and treated with or without 300 m-units of bSMase for 1 h at 37 °C. Mitochondrial fraction (5 μg of protein) was then incubated with 25 μg of cytosolic fraction for the indicated times at 37 °C. At the end of incubation, the mitochondrial and cytosolic fractions were separated by centrifugation (5000 g for 15 min) and Bax translocation was evaluated by Western blotting as described in the Experimental section, but in the absence of SDS. Cyto, cytoplasm; Mito, mitochondria.