Fig. 1. RagA/B paralogues determine distinct mTORC1 responses.
a, Transcript levels of the Rag isoforms in healthy human tissues (gtexportal.org). n, biological replicates. b, Western blot for RagA, RagB and RagC in mouse tissues. Calnexin is the loading control. The experiment was repeated once. c,d, Domain organization of the Rag isoforms. Numbering indicates amino-acid positions in the human sequence. Percentages represent similarity of each domain between Rag paralogues. Ex4 is the sequence encoded by exon 4 of the Rragb gene. e–h, S6K1, TFEB and 4EBP1 phosphorylation in control or RagABKO cells stably transfected with a control protein (FLAG–metap2) or with the indicated Rag isoforms. Cells were incubated in amino-acid-rich medium or starved of amino acids for 30 min: representative example (e) and quantification of three independent experiments, with unstarved control cells set to 1 (f–h). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. Two-way ANOVA and Sidak’s post-hoc test. i,j, S6K1 phosphorylation upon loss of RagA, RagB or both: representative example (i) and quantification of three independent experiments, with control cells set to 1 (j). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. One-way ANOVA and Tukey’s post-hoc test. k–n, RagA (k and l) but not RagB (m and n) loss causes persistent mTORC1 activity. Cells were incubated in amino-acid-rich medium, starved of amino acids for 1 h, or starved for 1 h and re-stimulated with amino acids for 15 min (addback, ‘ab’): representative examples (k and m) and quantification of three independent experiments, with unstarved control cells set to 1 (l and n). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. Two-way ANOVA and Sidak’s post-hoc test. o–p, The elevated mTORC1 activity in RagAKO cells upon amino-acid removal cannot be rescued by stable overexpression of the RagB isoforms. Cells were incubated in amino-acid-rich medium or starved of amino acids for 30 min: representative example (o) and quantification of three independent experiments, with unstarved control cells set to 1 (p). Bar height indicates average, error bars represent standard deviation; n = 3 biological replicates. Two-way ANOVA and Sidak’s post-hoc test. −aa, amino-acid-free DMEM + 10% dFBS. +aa, −aa medium supplemented with 1× amino acids. Exact P values are shown in the graphs. Source numerical data and unprocessed blots are available in source data.