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. 2022 Sep 12;24(9):1407–1421. doi: 10.1038/s41556-022-00977-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 9 — (a, b) Coomassie staining of RagB^long (a) or RagA (b) purified from RagABKO HEK293T cells as dimers with FLAG-tagged RagC^S75N (1 μg dimer per lane). The experiment was repeated once. (c, d) GTPase activity of RagA or RagB^long proteins dimerized to RagC^S75N (1 μM) and mixed in solution with the indicated concentrations of GATOR1. RagB^long was purified from mammalian cells and RagA was purified from either (c) bacteria or (d) mammalian cells. Data from three (c) or two (d) independent experiments were plotted and a curve was fit with non-linear regression analysis following a hyperbola model. Circle=average (c) or individual replicates (d), error bars=standard deviation. (e, f) in vitro interaction of purified Rag dimers with GATOR1 (containing DEPDC5^Y775A) in the presence of GDP. (e) Representative example. (f) Quantification of 3 independent experiments normalized for the background in the empty beads control. Bar Height=average, error bars=standard deviation, n = 3 replicates. One-way ANOVA and Tukey’s post-hoc test. (g) Quantification of the total RagA/B protein levels in Fig. 5a-i, with the RagA condition set to 1. Line=average, error bars=standard deviation, n = 9 biological replicates. One-way ANOVA and Tukey’s post-hoc test. (h-i) S6K1 phosphorylation in RagABKO cells transiently transfected with RagA, RagA and RagB^short, or all three RagA/B isoforms during starvation (amino-acid free DMEM + 10% dFBS) for the indicated time points. (h) Representative example. (i) Quantification of six independent experiments, with RagA-expressing cells at time point 0 set to 1. Circle=average, error bars=standard deviation, n = 6 biological replicates. Two-way ANOVA and Tukey’s post-hoc test. (j-m) Amino-acid titration in Neuro-2a cells knockout for RagA (j-k) or RagB (l-m) treated with the indicated amino-acid concentrations for one hour. (j,l) Representative examples. (k.m) Quantifications of 3 independent experiments, with unstarved control cells set to 1. Circle=average, error bars=standard deviation, n = 3 biological replicates. Two-way ANOVA and Tukey’s post-hoc test. Exact p values are shown in the graphs. n.s., not significant. Source numerical data and unprocessed blots are available in source data.

Source data