Extended Data Fig. 6 | Addition of a TOS motif to a Rag-binding-def icient
TFEB mutant rescues its phosphorylation and subcellular localization.
a, HeLa cells transiently expressing the cDNAs described in Extended Data
Fig. 5 were starved of amino acids for 60 min and restimulated with amino acids
for 30 min, in the presence or absence of 250 nM torin, and analysed by
immunoblotting using the indicated antibodies (replicated three times).
b, Cells described in a were either starved of amino acids for 60 min or starved
and restimulated with amino acids for 30 min, in the presence or absence of
torin, and analysed for TFEB subcellular localization by immunof luorescence
(replicated twice). c, Representative immunoblotting and quantification
(mean ± s.e.m.; n = 3) of HeLa cells stably expressing GFP–TFEB or
GFP–TOS-D30TFEB. Cells were either kept fed, starved of amino acids (−aa) or
serum (−FBS) for 2 h. d, Cells described and treated as in c were analysed by
immunof luorescence to assess TFEB subcellular localization (replicated three
times). Scale bar, 10 μm. e, Analysis of TFEB localization performed using a
dedicated script (Columbus software; Perkin-Elmer) that calculates the ratio
value resulting from the average intensity of nuclear TFEB–GFP f luorescence
divided by the average of the cytosolic intensity of TFEB–GFP f luorescence.
Results are mean ± s.e.m. P values were calculated on the basis of mean values
from 3 or 4 independent fields (Sidak’s multiple comparisons test).
***P < 0.0001; NS, non-significant (fed, P = 0.9989; −aa, P = 0.0946).