Figure 3.

Regulation of endogenous TFEB by mTOR and Rags. (A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin. (B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3. (C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A. (D) HeLa cells were starved in serum- and amino acid–free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB. (E) HeLa cells were incubated in normal medium (control) and serum- and amino acid–free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets. (F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione–Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.