Fig. 2. RagBshort and RagBlong are resistant to GATOR1.
a,b, RagABKO cells expressing each RagA/B isoform were transiently transfected with increasing amounts of GATOR1 plasmids (5 ng, 25 ng or 100 ng of each GATOR1 subunit) or metap2 (100 ng) as negative control: representative example (a) and quantification of four independent experiments, with RagA-expressing cells transfected with metap2 set to 1 (b). Bar height indicates average, and error bars represent standard deviation; n = 4 biological replicates. Two-way ANOVA and Tukey’s post-hoc test. c,d, Control and RagAKO cells were transiently transfected with high (200 ng) levels of each GATOR1 subunit or metap2 as negative control and treated with amino-acid-rich medium or starved of amino acids for 30 min before lysis: representative example (c) and quantification of three independent experiments, with unstarved metap2-transfected control cells set to 1 (d). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. Two-way ANOVA and Sidak’s post-hoc test. e, Schematic representation of the two binding interfaces of GATOR1 to RagA/B. f–h, Rag interaction with the inhibitory interface (depicted in f) is assessed by co-immunoprecipitating the whole GATOR1 complex: representative example (g) and quantification of three independent experiments, with the inactive mutant of RagA set to 1 (h). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. One-way ANOVA and Tukey’s post-hoc test. i–k, Rag interaction with the GAP interface (depicted in i) is assessed through co-IP with the Nprl2/3 dimer in DEPDC5KO cells: representative example (j) and quantification of three independent experiments, with the inactive mutant of RagA set to 1 (k). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. One-way ANOVA and Tukey’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. NS, not significant. Source numerical data and unprocessed blots are available in source data.