Extended Data Fig. 7: Characterization of the lysosomal localization of GATOR1, GATOR2, KICSTOR, and ILF3i1.
From: Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing
a, Wild-type, NPRL3 KO, or SZT2 KO HEK293T cells stably expressing GFP-WDR24 were starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min. Cells were then immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. b, Wild-type, MIOS KO, or NPRL3 KO HEK293T cells stably expressing GFP-C12orf66 were treated as in a, immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. c, Quantification of the colocalization of GFP-WDR24 (a) or GFP-C12orf66 (b) with LAMP2 using Pearson’s correlation coefficient. Quantification was carried out on 40 cells examined over 3 independent experiments. Data are mean ± s.d. n.s., not significant (unpaired two-sided Student’s t-test). d, Wild-type, MIOS KO, NPRL3 KO, or SZT2 KO HEK293T cells stably expressing 3×HA-tagged TMEM192 were subjected to an immunoprecipitation-based lysosome capture process. The WCLs and immunoprecipitated lysosome fractions (LysoIPs) were immunoblotted to detect the indicated proteins. Quantification was performed with ImageJ. Data are mean ± s.d. (n = 3 biologically independent experiments). Exact P values are shown in the graph (unpaired two-sided Student’s t-test). Note that all blots in the sgMIOS group were normalized with their corresponding wild type group. Source numerical data and unprocessed blots are available in source data.
