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. Author manuscript; available in PMC: 2015 Sep 26.
Published in final edited form as: Nature. 2015 Jan 7;519(7544):477–481. doi: 10.1038/nature14107

Extended Data 9. Stable expression of SLC38A9 mediates sustained mTORC1 activation upon amino acid starvation.

Extended Data 9

a. SLC38A9- or METAP2-stably expressing HEK293T cells were starved for the indicated time in medium without amino acids and serum. Cell lysates were analysed by immunoblot. Results are representative of two independent experiments (n=2) b. Representative images in the GFP channels of HEK293T cells stably expressing EGFP-LC3B and SLC38A9 or METAP2 starved for 120 min (related to Fig 4 B). Scale bar, 40 μm c. HEK293T cells stably expressing TFEB-STHA and SLC38A9 or METAP2 were starved for the indicated time. Cytoplasmic and nuclear fraction were analysed by immunoblot. Results are representative of two independent experiments (n=2). d. Immunoblot analysis of HEK293T cells stably expressing the indicated SLC38A9 constructs. e. SLC38A9 (S)- or METAP2 (M)-stably expressing HEK293T were starved for 50 min and then stimulated with amino acids for 20 min. Where indicated, cells were treated with ConcanamycinA (5μM) or DMSO during both incubation time. Cell lysates were analysed by immunoblot with the indicated antibodies. Results are representative of two independent experiments (n=2) f. SLC38A9 (S)- or METAP2 (M)-stably expressing HEK293T were treated for 30 min with DMSO (D), ConcanamycinA (C, 5μM) or Torin 1 (T, 250 nM) and then starved for the indicated times in presence of the inhibitors. Cell lysates were analysed by immunoblot. Results are representative of two independent experiments (n=2).