Inhibition of the PI-3-kinase signaling pathway induces partial development of competence in NGF-maintained sympathetic neurons. A, Bax-deficient sympathetic neurons were maintained in NGF (+NGF), deprived of NGF (−NGF), or maintained in NGF in the presence of a 50 μm concentration of the PI-3-kinase inhibitor LY294002 (+NGF+LY) for 36 hr. Bax deficiency permits these neurons to be deprived of NGF or to be treated with LY294002 yet remain alive to allow for the microinjection experiments. Neurons were then microinjected with cytochrome c (10 mg/ml) and rhodamine dextran (C) or rhodamine dextran alone (R), and the number of cells injected was determined as described in the legend to Figure 1. Results are the means ± SEM for three experiments with ∼100 cells counted for each time point per experiment. B, A 50 μm concentration of LY294002 inhibits the PI-3-kinase signaling-mediated phosphorylation of its substrate Akt in NGF-maintained sympathetic neurons. Western immunoblots show lysates of NGF-maintained sympathetic neurons either alone or in the presence of 50 μm LY294002 for 36 hr that were probed with an anti-phospho-specific Akt (α p-Akt) antibody to assess the inhibition of the PI-3-kinase signaling in neurons. Lysates were also probed with an anti-tubulin (α Tubulin) antibody as a loading control.