Fig. 5.

TFEB undergoes mTOR-dependent nuclear phosphorylation. a HeLa cells stably expressing TFEB-GFP were either starved of amino acids (aa) for 60 min, or starved and restimulated with amino acids for 30 min. Where indicated, cells were pretreated with 5 nM leptomycin B (Lepto) during starvation, prior to refeeding in the presence or absence of leptomycin B or Torin (250 nM). Upon treatment, cell extracts were analyzed by western blotting with the indicated antibodies. b Representative images of cells treated as in a and analyzed for TFEB localization by confocal microscopy. Scale bar: 10 μm. c HeLa cells were transfected with wild-type or M144A TFEB for 24 h. Cells were then either starved for 1 h or starved and restimulated with nutrients for 30′. Cell extracts were analyzed by western blotting with the indicated antibodies. d HeLa cells transfected with either ΔNLS-TFEB-GFP or wild-type TFEB-GFP were either starved for 60 min or starved and restimulated with amino acids for 30 min in the presence or absence of 250 nM Torin, and evaluated for TFEB phosphorylation by immunoblotting. e HeLa cells stably expressing TFEB-GFP were either starved for amino acids (aa) for 60′, or starved and restimulated with aa for 30′ in the presence or absence of inhibitors of mTOR (Torin; 250 nM), ERK (U0126; 10 μM), and GSK3 (SB415286; 50 μM). Cell extracts were analyzed by western blotting with the indicated antibodies