FIGURE 4.

Perturbation of mTORC1 regulation alters system dynamics, in accordance with model predictions. A, stable shRNA-expressing cell lines were established to perturb key regulatory proteins in the Akt/mTORC1 axis: TSC2, PRAS40, and Rheb. For each target, two hairpins targeting different sequences were selected, based on the extent of protein knockdown (% KD, as indicated), along with a non-targeting control line (shNEG). Representative immunoblots and quantification of target protein knockdown and S6K1 phosphorylation (p) for the indicated PDGF stimulation conditions are shown. Phospho-S6K1 data are normalized by total S6K1 and then by the mean value of each shNEG time course. The normalized data are reported as mean ± S.E. (n = 3 independent experiments). B, the corresponding model predictions are the means of the model ensemble (n = 10,000) for each condition, normalized in the same manner as the data. C, model predictions as in B, using an alternative parameter set ensemble acquired by fitting the data as in Fig. 3B but with the parameter constraints α = β = 1.