Extended Data Fig. 7: Phosphorylation of S6K by non-lysosomal mTORC1 responds to exogenous AAs and is regulated downstream of mTOR and growth factor signalling.
From: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources
(a) Immunoblots with lysates from WT and RagA/B KO HEK293FT cells, treated with media containing (+AA) or lacking AAs (–AA), or with Torin1 as shown, probed with the indicated antibodies. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (–AA), culture media were replaced by starvation media 1 h before lysis. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. n = 3 independent experiments. (b) As in (a), but for WT and RagC/D HEK293FT cells. n = 2 independent experiments. (c) AA starvation time-course (1-8 h) in WT and RagA/B KO HEK293FT cells shows that the residual S6K phosphorylation in the KOs at the early time points of starvation disappears at slightly later times. Starvation was performed as in (a). n = 3 independent experiments. (d) Immunoblots with lysates from WT and RagA/B KO HEK293FT cells, transiently transfected with siRNAs targeting TSC2 or a control RNAi duplex (siCtrl), probed with the indicated antibodies. n = 2 independent experiments. (e) Immunoblots with lysates from WT and RagA/B KO HEK293FT cells, treated with Akt inhibitor (AKTi; 10 μM, 30 min) as shown, probed with the indicated antibodies. n = 3 independent experiments. (f) Growth factor removal and re-addition is sensed similarly in RagA/B KO and WT MEFs. Cells were starved for 1 h in media lacking FBS (–) and then re-stimulated with insulin (+Ins) for 30 min. n = 4 independent experiments. (g) Rag KO cells are deficient in sensing glucose depletion, in line with previous studies. WT and RagA/B KO HEKs were cultured under basal glucose- and AA-replete conditions, or starved with media lacking glucose or AAs for 1 h (–), or first starved and then re-stimulated (–/+) with glucose or AAs for 30 min. n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. Unprocessed blots are available in source data.
