FIGURE 6.

Osmotic stress enhances mTORC1 kinase activity. A and B, HEK293 cells transfected with FLAG-Raptor were serum-starved overnight and exposed to osmotic stress. mTORC1 was immunoprecipitated (IP) with FLAG antibody and the in vitro kinase assay was performed with purified 4E-BP1 and S6K, respectively. The bar graph at the bottom shows the quantification of 4E-BP1 and S6K1 phosphorylation, respectively. A. U., arbitrary units. p-4E-BP1, phospho-4E-BP1; p-S6K, phospho-S6K. C, HEK293 cells were pretreated with 100 nm calyculin A for 30 min and then exposed to 0.5 m sorbitol for the indicated times. The cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. D, HEK293 cells were pretreated with 100 nm calyculin A and 20 μm SP600125 or 20 nm rapamycin where indicated and then exposed to 0.5 m sorbitol for an additional 60 min. The cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.