Fig. 1

Src is necessary and sufficient for amino acid-mediated activation of mTORC1. a SH-SY5Y cells were treated with SRC shRNAs, starved, and stimulated with amino acids for 30 min. Lysates were probed with antibodies as indicated. Box plots represent SE of n = 3 independent experiments. *p < 0.05. N.S. represents “not significant”. b SH-SY5Y cells were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation prior to stimulation with amino acids at several time points. Lysates were probed with antibodies as indicated. c SH-SY5Y cells were starved and treated with vehicle (DMSO) or PP2 (10 μM), or rapamycin (600 nM) for the last 2 h of starvation and then stimulated with amino acids (30 min). Lysates were probed with antibodies as indicated. d SH-SY5Y cells were treated as in b but with a single 30 min amino acid stimulation prior to immunofluorescence labeling of endogenous LAMP2 (red) and mTOR (green). e SH-SY5Y cells were treated as in a and labeled and analyzed as in d. Bar indicates 60 μm. Representative cells are shown where yellow or orange pixels indicate co-localization in the merged images. In all images in d and e insets show selected fields that were magnified by a factor of 4. Scale bar indicates 60 μm. Quantified data represent mean co-localization (Mander’s coefficient) of mTOR and LAMP2 in at least 15 cells for each condition. Box plots represent SE of n = 3 independent experiments. ** indicates significant differences (**p < 0.01) between all conditions in each group. f SH-SY5Y cells, transiently transfected with Y530F-Src, SrcΔSH3, or SrcΔSH2, were starved with amino acids (kept in dialyzed serum for 4 h). Cells were then lysed and immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states. g SH-SY5Y cells were transiently transfected with the indicated Src constructs and starved as in f. Cells were treated with PP2 (10 μM), where indicated, for the last 2 h of starvation prior to lysis and immunoblot analyses. GAPDH was used as a loading control in all immunoblot assays. Statistical differences between groups in a, d, and e were determined using ANOVA with Tukey’s post hoc test