Extended Data Figure 5: SZT2 is localized on the lysosome, which together with GATOR1 controls lysosomal localization of WDR59.
a, HEK293T or SZT2Flag/− 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, SZT2Flag/− cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where indicated. Cells were immunostained with anti-Flag (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 ± s.d. with 100 cells analysed for each condition. g, DEPDC5-deficient HEK293T cells were deprived of amino acids for 1 h and stimulated with amino acids for 20 min where 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 where indicated. Total cell lysates were analysed by immunoblotting. i, mRNA levels of DEPDC5 in control and DEPDC5-deficient HEK293T cells were measured by qPCR and normalized to β-actin. AU, arbitrary units. 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 where indicated. The localization of WDR59 and LAMP2 was determined by immunostaining. Data (a, b, g–j) are representative of three independent experiments.
