Extended Data Figure 2: SZT2 forms a complex with GATOR.
a, Mass spectrometry analysis of SZT2-interacting proteins in HEK293T cells stably overexpressing Flag–RAP2A or Flag–SZT2. Specific recovery of the GATOR complex components is shown. b, c, A diagram of the template (b) and sequence (c) used to introduce a Flag tag into the SZT2 locus. d, Total cell lysates from HEK293T cells, HEK293T cells stably expressing Flag–SZT2, or SZT2Flag/− cells were analysed by immunoblotting. e, Control or SZT2Flag/− cells were deprived of amino acids for 1 h and stimulated with amino acids for 10 min where indicated. Total cell lysates were analysed by immunoblotting. f, Size-exclusion chromatography of the SOG complex purified from SZT2Flag/− cells on a Superose 6 Increase 10/300 GL column. The insert shows the calibration of the column, and the equation used for calculating the size of the SOG complex. g, Peak fraction of SOG from f was analysed by silver staining and immunoblotting. h, Sucrose density gradient centrifugation analysis of the sedimentation pattern of GATOR components from control or SZT2-deficient HEK293T cells. Dashed box indicates fractions containing LAMP1, SZT2, WDR59, WDR24, NPRL3 and NPRL2. Data (a, d–h) are representative of three independent experiments.
