Figure 2. Differential feedback activation of Akt and ERK phosphorylation by rapamycin and KU63794 in MiaPaCa-2 cells.

Cultures of MiaPaCa-2 cells were incubated in the absence (−) or in the presence of KU63794 (Ku) at 1 µM or 5 µM or rapamycin (Rap) at 10 or 100 nM for 2 h in DMEM containing 5 mM glucose, as indicated. Then, the cells were stimulated for 2 h with 5 nM neurotensin (NT) and 10 ng/ml insulin (Ins) and lysed with 2×SDS–PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies that detect the phosphorylated state of S6K at Thr³⁸⁹ (pS6K), S6 at Ser^235/236 (pS6), 4E-BP1 at Thr^37/46 and Thr⁷⁰, Akt at Ser⁴⁷³ and ERK at Thr²⁰² and Tyr²⁰⁴. Immunoblotting with antibodies that recognize total S6K, S6, 4E-BP1, Akt and ERK was used to verify that the cell treatments did not change the total level of these proteins and confirm equal gel loading. Fold increase in ERK phosphorylation was quantified using Multi Gauge V3.0 and plotted as bars. Similar results were obtained in 3 independent experiments.