EGF prevents BAD but not BAX translocation to the mitochondria. (a) LNCaP cells were plated in 100 mm dish for 2 days before either control media (10% serum) or serum-free media (no serum) was added for 20 h. Cells were harvested at this point (0 h) (lane 1–4) for subcellular fractionation. Additionally, serum-starved cells were further treated with 10% serum-containing medium (serum), LY in 10% serum-containing medium (LY+serum), LY in serum-free medium (LY) or LY in serum-free medium and EGF (LY+EGF) for 90 min before harvesting for subcellular fractionation (lane 5–12). Fractionation was performed as in Materials and methods and equal protein amount for cytosol (C) and mitochondria (M) fractions were subjected to SDS–PAGE and immunoblot analysis. Purity of the fractions was determined by blotting for cytosol- or mitochondria-specific protein, Cu/Zn SOD and VDAC, respectively. (b and c) densitometry analysis of the amount of BAD and BAX translocation into the mitochondria. Results are shown as the ratio of mitochondrial BAD or BAX over VDAC converted to % of the ratio obtained in cells incubated with serum (% of relative intensity). (d) Activation of BAX was assessed as described in Materials and methods following cell treatment as described above. Results shown in (a and c) represent one representative experiment of at least three.