HAP-1 cells expressing RAPTOR-GFP were set up for parallel experiments: live imaging to reveal RAPTOR dynamics (B, D, E, F) and immunoblotting to reveal S6K phospho T389 (A, C) and ULK1 phospho S757 (A). Immunoblotting was for the indicated times after re-stimulation with a mixture of MEM and NE amino acids. The linearity of the detection for S6K phospho T389 is shown for ascending amounts of lysate (last two blots in A). In the experiment shown in A, re-stimulation was in triplicate. In the blot shown in C, concanamycin A was added to a final concentration of 2 µM for the indicated samples. In order to obtain graphs of RAPTOR translocation to punctate structures (B and D) we examined many cells for each condition coming from 3 to 4 independent imaging experiments (13 cells for control and 137 cells for aa’s in panel b; 59 cells for aa’s, 77 cells for aa’s + EGF + Ins and 60 cells for aa’s + conc in panel d). The number of puncta upon stimulation was counted in each cell using the Spots Detection function of Imaris software (Bitplane/Andor), and the average of all cells in each condition together with the standard deviation is plotted here. The number of cells responding to the various treatments and expressed as a percentage is shown in panels E and F, together with the statistical significance of the differences as indicated. Significance was evaluated using one-way ANOVA with a Bonferoni post-hoc test. Error bars represent standard error of the mean. (G) HAP-1 cells expressing RAPTOR-GFP were kept in fed conditions, starved for 60’ or starved for 60’ and re-stimulated with amino acids and growth factors for 5’, 10’, 20’ and 30’. The RAPTOR-GFP protein was immunoprecipitated from these cells and the immunoprecipitates were analysed for a variety of potential interacting proteins as shown. All experiments were conducted in 0.3% CHAPS lysis buffer with the exception of the one labelled RAGA* which was conducted in 0.3% deoxy big CHAP.