Neither PI-3-K nor MEK/MAPK is required for NGF-dependent inhibition of Bax translocation from cytoplasm to mitochondria. A, This schematic diagram depicts the treatment paradigm used in B. Sympathetic neurons were deprived of NGF for 18 hr before a 30 min incubation with LY294002 (50 μm), PD98059 (25 μm), or vehicle alone (DMSO, 0.1%). Neurons were next washed and treated with NGF (300 ng/ml) in the continued presence of LY294002, PD98059, or DMSO for an additional 12 hr. B, Sympathetic neurons treated as inA were homogenized in an isotonic buffer and subcellular fractions produced by differential centrifugation. Equal volumes of the cytosolic (Cyto) fractions and heavy membrane (HM) fractions containing mitochondria were subjected to Western analysis by using antibodies directed against Bax, cytochrome c, lactate dehydrogenase (LDH, a cytosolic protein), and cytochrome oxidase IV (COX IV, a mitochondrial protein). LDH and COX IV immunoblots demonstrate not only that the cytoplasmic and heavy membrane fractions do not contain significant amounts of contamination, but also that equal amounts of protein from each of these fractions were analyzed. This experiment was performed on two independent cultures with similar results.