Fig. 3: Function of the TFEB-nc-Rag GTPases interface.

a, Cells expressing wild-type (WT) or mutant TFEB-GFP were analysed using immunofluorescence to determine the percentage of cells showing nuclear TFEB, shown as mean ± s.e. throughout; n = 12 independent fields per condition. b, Immunoblot of HeLa cells expressing wild-type or mutant TFEB-GFP. c, Representative co-immunoprecipitation of Flp-In 293 T-REx cells transfected with wild-type or mutant TFEB-GFP. d, Microscopy analysis of Torin1-treated HeLa cells, n ≥ 5 independent fields per condition. ***P ≤ 0.0001 throughout. One-way analysis of variance (ANOVA), Dunnett’s multiple comparisons test. e, Representative co-immunoprecipitation of HeLa RagC KO cells transfected with the indicated constructs. f, Immunoblot of RagC KO HeLa cells transfected with empty vector or wild-type RagC or RagC(D294R). Cells were amino acid starved and refed in the presence or absence of 250 nM Torin1. g, Representative co-immunoprecipitation of HeLa RagA KO cells transfected with the indicated constructs. h, Immunoblot of RagA KO HeLa cells transfected with empty vector or wild-type RagA or RagA(H104D/Q107R/E111R). i–k, Cells as in f were analysed using immunofluorescence and the percentage of the cells were determined to show nuclear TFEB (i) (n = 5 fields per condition); TFEB–RagC colocalization (j) (n ≥ 5 fields per condition, ***P ≤ 0.0001, unpaired t-test) and mTOR–RagC colocalization (k) (n ≥ 12 fields per condition, unpaired t-test). l–n, Cells as in h were analysed using immunofluorescence and quantified to calculate the percentage of the cells showing nuclear TFEB (l) (n ≥ 4 independent fields per condition), TFEB–RagA colocalization (m) (n ≥ 4 fields per condition, **P ≤ 0.002, unpaired t-test) and mTOR–RagA colocalization (n) (n = 5 independent fields per condition, unpaired t-test). Scale bar, 10 μm. NS, not significant; aa, amino acid; Ctrl, control; HA, haemagglutinin; IP, immunoprecipitation; GST, glutathione S-transferase.