Extended Data Figure 5. SZT2 is localized on the lysosome, which together with GATOR1 controls lysosomal localization of WDR59.

a, HEK293T or SZT2^FLAG/− cells were immunostained with anti-FLAG together with anti-Lamp1. b, Control or WDR59-deficient HEK293T cells were immunostained with anti-WDR59 together with anti-Lamp2. c – e, SZT2^FLAG/− cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min when indicated. Cells were immunostained with anti-FALG (c – e), together with anti-Lamp2 plus anti-WDR59 (c), or anti-Lamp2 plus anti-EEA1 (d), or anti-Rab7 plus anti-PMP70 (e). f, Quantification of the co-localization among SZT2, PMP70, EEA1, Rab7, Lamp2 and WDR59. Data represent mean ± SD with 100 cells analyzed under each condition. g, DEPDC5-deficient HEK293T cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min when indicated. The localization of WDR59 and Lamp2 was determined by immunostaining. h, Control, SZT2-deficient, DEPDC5-deficient, and NPRL3-deficient cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min when indicated. Total cell lysates were analyzed by immunoblotting. i, mRNA levels of DEPDC5 in control and DEPDC5-deficient HEK293T cells were measured by qPCR and normalized to that of β-actin. AU, arbitrary unit. j, Control or SZT2-deficient HEK293T were treated with rapamycin (100 nM), deprived of amino acids for 1 h and stimulated with amino acids for 20 min when indicated. The localization of WDR59 and Lamp2 was determined by immunostaining. Data (a, b, g – j) are representatives of three independent experiments.