Fig. 3.

TFEB is subject to CRM1-mediated nuclear export. a Live cell steady state images of indicated cargo vectors stably expressed in MCF7 cells. n > 120 cells per condition. b Western blot of HT29 cells expressing doxycycline-inducible WT or indicated mutant TFEB-GFP (−Dox). c Fluorescence images of HT29 cells stably expressing indicated WT and mutant TFEB-GFP treated with Torin 1 (25 nM) for 1 h before cells were washed and placed in Torin 1-free medium for 15 or 60 min. n > 300 cells per condition. d Western blot using indicated antibodies to detect CRM1 and RAN after pull down of immobilized HIS-tagged TFEB (aas 1–200). All proteins were bacterially expressed and purified. Purified TFEB was visualized by Coomassie staining (lower panel). e Schematic depicting the role of CRM1 and RAN in nuclear export using TFEB as a potential substrate. Top: RAN-WT; Bottom, RAN-Q69L. See text for details. f Fluorescence images of steady state subcellular localization of MCF7 cells stably expressing the TFEB 1-159-NLS-cargo together with either mCherry-LacR or mCherry RAN-Q69L. Cells were treated with DMSO, Torin 1 (250 nM) alone or with LMB (20 nM) as indicated. Subcellular localization of TFEB-GFP was quantified for cells expressing mCherry. n > 100 cells per condition. In the RanQ experiment, only mCherry positive cells were counted. Scale bars = 20 μM. Error bars = SD. Student’s t-test; ****P < 0.0001, n.s. not significant