Research Paper Volume 2, Issue 8 pp 487—503

Figure 5. (A a Immunoblot analysis revealing increased phosphorylation of AkT/PKB on serine 473 under nutrient deprivation (upper panel). The relevant band is indicated by the arrow. Band quantization values (band volume) in band intensity (b.i.) units are indicated. The same filter was stripped and re-hybridized with an anti total AkT antiserum to ensure equal protein expression and sample loading (central panel); a lower strip of the same filter was hybridized with an antiserum specific for phospho S6 (lower panel, band indicated by arrow). Picture representative of several independent experiments. b Protein lysates from mock and mTOR-silenced cells grown under serum free DMEM with glucose and aminoacids were treated as in A. Relevant bands are indicated by arrows. Densitometry of p-AkT bands is reported. (B) a Effect of the PI3 Kinase inhibitor compound LY294402 on Phoenix cell survival in serum-free medium. Cells were incubated for 72 hours with or without glucose as indicated. The inhibitor or vehicle alone (DMSO, 1:500 final dilution) were added at time 0. Values are Mean ±SD of triplicate wells. Representative of three independent experiments. Note that lower concentrations of LY294002 had no effect on cell survival in either medium. b Immunoblot analysis of protein lysates from cells treated as in a and incubated for 24 hours. Phospho-AkT (serine 473) and phospho-S6 (serine 235-236) were detected by specific antisera. Relevant bands (the middle one within the triplet for AkT) are indicated by arrows; equal protein loading was verified by reversible Ponceau S staining.