Figure 1

Differential activation of mTORC1 and mTORC2 in PTEN^wt and PTEN^mu GBM cells along with downstream signaling. (a) Schematic representation of proposed hypothesis: role of PTEN in mTORC2 signaling pathways. (b–h) PTEN^mu and PTEN^wt cells were harvested and lysed and kept in phosphate-buffered saline (PBS) with protease and phosphatase inhibitors. Protein concentrations were measured by the Bradford’s protein assay reagent. Proteins (50-100 μg) from each sample were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 5–12%) followed by electrotransfer into nitrocellulose/polyvinylidene difluoride (PVDF) membrane and probed with primary antibodies (1:1000–1:2000) and incubated overnight at 4 ^oC. Then the membrane was washed with Tween-20 (0.1%) containing Tris-buffered saline (TBS) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000). They were washed and target proteins were identified by the West Pico chemiluminiscence detector system or Biorad ChemiDoc MP System (Bio-Rad). Developed bands of corresponding proteins were densitometrially analyzed by ImageJ software. (b) Level of PTEN in PTEN^mu and PTEN^wt cells. (c) Status of mTOR, p-mTOR Ser2448, p-mTOR Ser2481, Rictor and Raptor. (d) AKT, the downstream substrate of mTORC2 was highly phosphorylated at Ser473 in PTEN^mu cells. (e) Status of AKT phosphorylation at the Thr450 and Thr308 positions. (f) Densitometric analysis showed phosphorylation of AKT at the Thr450, Thr308 and Thr 473 sites. Values were normalized against the relative expression of β-actin and total AKT, quantified by ImageJ software. Each value was the mean±s.d. of three independent experiments. *P<0.05, significant difference between two test groups. (g) Status of Ser9 phosphorylation of GSK3β, the substrate of activated AKT. (h) mTORC1 activity readout as identified by phosphorylation of S6K1 and 4E-BP1. β-Actin served as loading control for all the western blots.