Fig. 3. Lysosomal V-ATPase increases in response to mTORC1 inactivation.

a Magnetic enrichment of lysosomes from MEFs, analysed by western blot. PNS: post nuclear supernatant; FT: column flow through; Elu: lysosomal eluate. Contaminating organelle markers are Golga1 (Golgi), Calreticulin (ER), Vdac (mitochondria), Pex19 (peroxisomes). b Enriched proteins in lysosomal fractions, quantified by label-free mass spectrometry. The dashed line (log₂ fold change >2) demarcates the cut-off for lysosomal proteins. c Changes in lysosomal proteins after 1 h aa starvation + 30 min aa restimulation (+aa) versus 1 h aa starvation (−aa) in MEFs, quantified by label-free mass spectrometry. d V-ATPase subunits in membrane fractions of dextran-loaded lysosomes from MEFs after 1 h ± torin 1 [400 nM], or after 1 h amino acid starvation ± 30 min aa restimulation (±aa), analysed by western blot. e V-ATPase subunit abundance changes in membrane fractions, quantified by western blot for torin 1/control and +aa/−aa as in d. Data are normalized replicate mean ± SEM. Dashed lines indicate protein abundance in the control/−aa groups. a One representative of n = 5 biologically independent experiments. b, c n = 5, e n = 5 (V1A, V1E, Vod1 + torin 1, V1E + aa) or n = 9 (V1A, Vod1 +aa) biologically independent experiments. In b, c, adjusted p-values were calculated using limma moderated t-statistic and adjusted with the Benjamini–Hochberg method for multiple testing. In e, p-values were calculated using a one sample t-test with a hypothetical mean of 1. Source data are provided as a Source data file.