Fig. 1.

Effect of PP242 catalytic and RAD001 allosteric rapalog inhibitors on mTOR downstream effector protein phosphorylation and protein synthesis in endometriod cancer cells in culture. (A) AN3CA cells in tissue culture were treated for 5 h or 10 h with 2.5 µM PP242 or 100 mM RAD001, considered maximal inhibitory doses based on IC₅₀ studies in a variety of cell types. Cells were lysed, equal amounts of soluble cell protein extracts resolved by SDS-PAGE and analyzed by protein immunoblotting with specific antibodies as shown. Phosphorylation immunoblotting of proteins was determined by immunoblotting membrane first with phospho-specific antibody then stripping the membranes followed by re-probing with non-phospho-specific antibodies. ECL was used for detection. Results shown are representative of three independent experiments. (B) Protein synthesis rates were determined 5 h after treatment of AN3CA cells with 2.5 µM PP242 or 100 mM RAD001, by [³⁵S]-methionine incorporation. Standard activity of label incorporation into nascent protein was determined by TCA precipitation and liquid scintillation counting. Results were normalized to control untreated cells. Standard errors of the mean (SEM) shown. *, P<0.05 by t-test.