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. 1999 Sep 6;146(5):955–966. doi: 10.1083/jcb.146.5.955

Figure 8.

Figure 8

The Ras–PI3-kinase–Akt pathway is required for the synergistic survival observed with NGF and KCl together. a, Ras activity is necessary for ∼50% of synergistic survival. NGF-selected sympathetic neurons were infected with 200 MOI of dnRas or GFP adenovirus, and one day later were switched into 5 ng/ml NGF (5N) plus or minus 10 mM KCl (10K). Neuronal survival was monitored two days later by MTT assay. Results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. *Indicates those results that are significantly different (P < 0.01) from 5 ng/ml NGF plus 10 mM KCl. b, Dominant-negative Ras partially inhibits Akt and ERK phosphorylation induced by NGF plus KCl. Western blot analysis for Akt and ERK phosphorylation in sympathetic neurons infected with 200 MOI dnRas adenovirus and then maintained in 5 ng/ml NGF (5N) and/or 10 mM KCl (10K). Lysates were analyzed with antibodies specific for serine-phosphorylated Akt (P-ser Akt), tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERKs), or total Akt or ERK protein. All samples were normalized for equal amounts of protein. All ERK panels are reprobes of one blot, and Akt panels reprobes of another. c, PI3-kinase is necessary for all of the survival seen with NGF plus KCl. MTT assays of sympathetic neurons maintained in various concentrations of NGF (ng/ml) and/or KCl (mM) treated coincidently with 100 μM LY294002 (LY) and/or 75 μM PD98059 (PD). Results represent data obtained from one representative experiment performed in triplicate, and each point is the mean ± SD. Similar results were obtained in three separate experiments. *Indicates those values that are significantly different from 5 ng/ml NGF plus 10 mM KCl (*) or 10 ng/ml NGF plus 10 mM KCl (**). d, Akt activity is necessary for all of the survival seen with NGF plus KCl. NGF-selected sympathetic neurons were infected with 10–100 MOI dnAkt adenovirus and, one day later, were switched to 5 ng/ml NGF plus 10 mM KCl. All neurons infected with dnAkt adenovirus were also infected with 50 MOI of an adenovirus expressing the TTA transactivator. Control neurons were infected with 100 MOI of the TTA adenovirus alone. Results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. *Indicates those values that are significantly different (P < 0.01) from 5 ng/ml NGF plus 10 mM KCl.