Extended Data Fig. 8 | TFEB phosphorylation and cytosolic retention
requires active RagC/D. a, RagC-knockout HeLa cells were transfected with
RR AGD-targeting siRNA (siRagD) for 72 h, then either starved of amino acids for
60 min or starved and restimulated with amino acids for 30 min and analysed
by immunoblotting using the indicated antibodies (replicated three times).
b, FLCN-knockout and control HeLa cells overexpressing the TFEB–GFP
construct 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 250 nM
torin, and then analysed by immunoblotting with the indicated antibodies
(replicated three times). c, Immunof luorescence analysis representative of
triplicate experiments of TFEB in FLCN-knockout and control HeLa cells kept in
amino acid deprived medium (−aa) or restimulated in amino-acid-containing
medium (+aa). Scale bars, 10 μm. d, FLCN-knockout HeLa cells transfected with
empty vector or with constitutively active RagD (RagD(S75L)) were either
starved of amino acids for 60 min, or starved and restimulated with amino
acids for 30 min, and analysed by immunoblotting with the indicated
antibodies (replicated three times). e, f, FLCN-knockout Hela cells transfected
with empty vector or constitutively active RagC (RagC(S75L)) (d) or
constitutively active RagD (RagD(S77L)) (e) and kept in basal medium were
immunostained with the indicated antibodies (replicated three times). Scale
bars, 10 μm.