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. 2000 Oct 1;20(19):7228–7237. doi: 10.1523/JNEUROSCI.20-19-07228.2000

Fig. 4.

Fig. 4.

PI-3-K regulates NGF-dependent metabolism of sympathetic neurons. A, Sympathetic neurons were treated for 3 min with medium alone, medium containing NGF (100 ng/ml), or pretreated with LY294002 (50 μm) or Wortmannin (1–10 μm) for 30 min and then treated with NGF in the continued presence of the respective PI-3-K inhibitor. Detergent extracts were produced, and lipid kinase assays (top panel) were performed on phosphotyrosine immunoprecipitates. P-Akt (S473 or T308) immunoblotting was performed on the supernatants from the lipid kinase assays (middle panels), and α-tubulin immunoblotting (bottom panel) was also performed to confirm equal protein loading. This experiment was performed twice with identical results, and the bar graph represents the means. B, Cultures of sympathetic neurons were treated with NGF (50 ng/ml) in the presence of increasing concentrations of LY294002 for 2 d. After this time, detergent extracts produced from these cultures were subjected to P-Akt (S473) immunoblotting (top panel). Equal protein loading was confirmed by reprobing the same immunoblot with antibodies directed against α-tubulin (bottom panel). C, Sympathetic neurons were treated with NGF (50 ng/ml), vehicle (DMSO, 0.1%), anti-NGF in the presence of BAF (50 μm), or NGF (50 ng/ml) with LY294002 (50 μm) and BAF (50 μm) for 2 d. The neurons were fixed and subjected to toluidine blue staining as described in Materials and Methods. Somal diameters of 100−120 neurons were determined from each condition from three independent cultures. Error bars represent SEM. The somal diameter of neurons in NGF medium was smaller than that normally observed because of the fixation, staining, and dehydration of the neurons. D, Sympathetic neurons were treated with NGF containing vehicle alone (DMSO, 0.1%), NGF with LY294002 (50 μm), NGF with LY294002 and BAF (50 μm), or anti-NGF with BAF for 2 d. Cultures were then subjected to MTT analysis as described in Materials and Methods. The data are presented as the percentage of MTT reduction compared with NGF-maintained neurons, and each condition was performed in quadruplicate in four independent cultures. Error bars represent SEM.