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. Author manuscript; available in PMC: 2012 Jan 1.

Published in final edited form as: Mol Cancer Res. 2010 May 25;8(6):896–906. doi: 10.1158/1541-7786.MCR-09-0409

Regulation of the rictor T1135 phosphorylation. A. The phosphorylation of rictor on the Thr-1135 site is mTOR dependant. MDA-MB-435 cells were infected with lentiviruses expressing shRNA targeting luciferase (control) and mTOR. Following a serum starvation for 24 hrs and stimulation by IGFI (40 ng/ml) for 30 min. The cells were lysed and the equal amount of protein lysates were analyzed by western blotting by the indicated antibodies. B. The ILK knockdown inhibits the phosphorylation of rictor on the Thr-1135 site. MDA-MB-435 cells were infected with lentiviruses expressing shRNA targeting luciferase (control) and ILK. Following a serum starvation for 24 hrs and stimulation by IGFI (40 ng/ml) for 30 min. C. The growth factor-dependent phosphorylation of rictor is prevented by inhibition of PI3K. The MDA-MB-435, A549 and Hela cells were serum starved for 24h. Where indicated, cells were pre-incubated or not with the PI3-kinase inhibitor LY294002 at 50 mM concetration for 1 hour and stimulated with IGF-I (40 ng/ml) for 30 min. D. The rictor phosphorylation is rapamycin dependent. The MDA-MB-435, A549 and Hela cell lines were grown in the growth medium containing 10% serum with or without rapamycin at 100 nM concentration for the indicated periods of time.

Regulation of the rictor T1135 phosphorylation. A. The phosphorylation of rictor on the Thr-1135 site is mTOR dependant. MDA-MB-435 cells were infected with lentiviruses expressing shRNA targeting luciferase (control) and mTOR. Following a serum starvation for 24 hrs and stimulation by IGFI (40 ng/ml) for 30 min. The cells were lysed and the equal amount of protein lysates were analyzed by western blotting by the indicated antibodies. B. The ILK knockdown inhibits the phosphorylation of rictor on the Thr-1135 site. MDA-MB-435 cells were infected with lentiviruses expressing shRNA targeting luciferase (control) and ILK. Following a serum starvation for 24 hrs and stimulation by IGFI (40 ng/ml) for 30 min. C. The growth factor-dependent phosphorylation of rictor is prevented by inhibition of PI3K. The MDA-MB-435, A549 and Hela cells were serum starved for 24h. Where indicated, cells were pre-incubated or not with the PI3-kinase inhibitor LY294002 at 50 mM concetration for 1 hour and stimulated with IGF-I (40 ng/ml) for 30 min. D. The rictor phosphorylation is rapamycin dependent. The MDA-MB-435, A549 and Hela cell lines were grown in the growth medium containing 10% serum with or without rapamycin at 100 nM concentration for the indicated periods of time.