Fig. 2: Amino acid deprivation induces Rap1 activity.

In a through (f), endogenous Rap1 activity (Rap1A+B-GTP) was assessed through immunoprecipitation of GTP-bound Rap1 and immunoblotting against the indicated proteins. Cells had been subjected to 1 h depletion of all amino acids (−AA) (a, e, f), glucose (b) or serum (c, e), and re-stimulated with amino acids for 30 min (a), or treated for 1 h with 20 nM Rapamycin (Rapa), 50 nM Torin1 or DMSO control (d). Lysates were prepared from serum deprived HEK293T cells (a), HEK293A cells (b–d), RagA/B knockout HEK293A cells (e) or MEF cells deficient in GCN2 (f). Graphs represent relative immunoblot band intensity from n experiments: a, b, c, e, f n = 3, d n = 4. In a, d, e, f Rap1-GTP and total Rap1 were processed on separate blots due to technical reasons. Statistical data are presented as mean values ± s.e.m. n.s. = not significant (P > 0.05); Student’s t-test; two-sided, unpaired. See Source Data File for statistics source data. Uncropped images of blots are shown in Supplementary Fig. 16.