Extended Data Fig. 3 | Rag GTPases are required for TFEB phosphorylation.
a, HeLa cells stably expressing GFP–TFEB were transfected with siRNAs
targeting both RR AGC and RR AGD or with a control siRNA (siCtrl). Seventy-two
hours after transfection, cells 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 by immunoblotting using the indicated
antibodies (replicated three times). b, Immunof luorescence analysis of TFEB
localization in HeLa cells stably expressing GFP–TFEB and transfected with
RR AGC- and RR AGD-targeting siRNA or with control siRNA (siCtrl) and after
48 h with empty vector (left), HA–RagC or HA–RagD (right). Cells were either
starved for amino acids for 60 min (−aa) or starved and then restimulated with
amino acids for 30 min (+aa) (replicated three times). Scale bar, 10 μm.