Rags are required for starvation-induced lysosomal recruitment of FNIP1. (A) Representative Western blots of anti-HA immunoprecipitation (IP) from WT HeLa cells and a HeLa line expressing 2×HA-FNIP1 from the endogenous locus. Molecular masses are given in kilodaltons. (B) Representative immunofluorescence experiment showing 2×HA-FNIP1 localization in fed and starved conditions ± siRNA-mediated RagC depletion. Bars, 10 µm. Insets are 7.3 µm wide. (C) Quantification of FNIP1 lysosome localization as defined by percentage of cells with five or more FNIP1 spots that are also LAMP1 positive (mean ± SEM; n = 3 experiments; 46–54 cells per condition; ****, P < 0.0001; ANOVA with Bonferroni’s post hoc test).