Fig. 5.

An interaction between TFEB and mTOR on the cytoplasmic surface of lysosomes. (A) Immunofluorescent staining showing the colocalization of TFEB-GFP and LAMP1 +/− torin 1 treatment (2μM, 2 hours). Cells were permeablized for 10 seconds with 0.1% saponin prior to fixation to extract the diffuse cytoplasmic pool of TFEB. This strategy facilitates visualization and quantification of the lysosomal signal for TFEB. (B) Quantification of the intensity ratios for LAMP1 and TFEB-GFP in cells treated +/− torin 1 (2 μM, 2 hours, n=3 experiments, average of 21,874 lysosome analyzed per condition/per experiment, * p<0.05, t-test). (C) Immunofluorescence images showing extensive colocalization of TFEB and mTOR on lysosomes following torin 1 treatment (2 μM, 2 hours). (D) Western blot of anti-GFP immunoprecipitations from control HeLa M cells versus a TFEB-GFP stable line +/− torin 1 pretreatment (2μM, 2 hours). (E) Western blots of anti-GFP immunoprecipitations from torin 1 treated cells that demonstrate the lack of interaction between Δ30TFEB-GFP, mTOR and raptor. (F) Detection of native TFEB following subcellular fractionation of Hela cells +/− torin treatment (2μM, 2 hours). Arrows indicate the relative positions of the phosphorylated TFEB (upper) and the dephosphorylated TFEB (lower). (G) Quantification of the abundance of nuclear TFEB in the preceding fractionation experiments (n=3 experiments, *p<0.05, t-test). All scale bars = 10 μm.