Fig. 6

Myotube mTORC1 signaling and protein synthesis. Cells were starved for 8 h in HEPES-buffered saline. Control was starved for an additional 70 min in fresh HEPES. “Leucine” was starved for 60 min in fresh Hepes and 10 min in fresh Hepes with leucine. “Chloroquine + leucine” was starved in fresh HEPES with chloroquine for 60 min and 10 min in fresh HEPES with leucine. A Phosphorylation of mTORC1 at Ser 2448 in control, leucine (1 mM for 10 min), and chloroquine + leucine (2 mg/ml chloroquine for 60 min followed by 1 mM leucine for 10 min) conditions. Insert shows representative western blot for each condition. B Phosphorylation of S6K1 at Thr 389 (same conditions). Insert shows representative western blot for each condition. Data are mean ± SEM. N = 8 for both treatment groups. *Different from control, P < 0.05. &Different from chloroquine + leucine. Chq, chloroquine. C Western blot analyses of protein synthesis using the SUnSET technique. Nutrient Rich-Control and Chloroquine were serum starved for 16 h and nutrient starved for 1 h (2 mg/ml for Chloroquine group only) prior to 30 min puromycin (1 µM) exposure. Nutrient Starve groups were starved under the same conditions as described above for mTORC1 signaling prior to 30 min puromycin (1 µM) exposure. Data are mean ± SEM, N = 6. ^abcColumns with uncommon letters differ, P < 0.05