Figure S4.
GABARAPs interact directly with NUFIP2 and G3BP1. (A) Immunoblot analysis of proteins associated with lysosomes purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from parental HeLa (WT), LC3TKO, GBRPTKO, and hexaKO cells treated with 4 mM LLOMe for 30 min. (B) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 deletions with GST or GST-tagged GABARAP (GABA). (C) GST pull-down assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-tagged GABARAP deletions. (D) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2/G3BP1 with GST or GST-tagged GABARAP mutants. (E) Immunoblot analysis of denaturing HA IP performed on untreated or 4 mM LLOMe treated (30 min) lysates from ATG8 knockout (hexaKO) or ATG4/ATG8 knockout (decaKO) HeLa cells expressing HA-GBRPL1-G. (F) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged GABARAP deletions. (G) Summary of interactions between GABARAP and G3BP1. (H) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged NUFIP2. (I) Quantification of Fig. S4 H. Data (% binding) represents the percentage of the corresponding protein relative to its input. (J) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-G3BP1. (K) GST pull-down assay of in vitro translated and radiolabeled GFP-tagged NUFIP2 with GST or GST-tagged G3BP1 deletions. (L) Immunoblot analysis of interaction between NUFIP2 and G3BP1 in HEK293T cells transfected with FLAG or FLAG-NUFIP2 with 2 mM LLOMe treatment for 30 min. (M) HCM images corresponding to Fig. 8 C. White masks, algorithm-defined cell boundaries. Yellow masks, computer-identified overlap of mTOR and LAMP2. Ctrl, control (untreated cells). AR, autoradiography. Data, means ± SEM; HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). **, P < 0.01, ANOVA. See also Figs. 7 and 8. Source data are available for this figure: SourceData FS4.

GABARAPs interact directly with NUFIP2 and G3BP1. (A) Immunoblot analysis of proteins associated with lysosomes purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from parental HeLa (WT), LC3TKO, GBRPTKO, and hexaKO cells treated with 4 mM LLOMe for 30 min. (B) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 deletions with GST or GST-tagged GABARAP (GABA). (C) GST pull-down assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-tagged GABARAP deletions. (D) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2/G3BP1 with GST or GST-tagged GABARAP mutants. (E) Immunoblot analysis of denaturing HA IP performed on untreated or 4 mM LLOMe treated (30 min) lysates from ATG8 knockout (hexaKO) or ATG4/ATG8 knockout (decaKO) HeLa cells expressing HA-GBRPL1-G. (F) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged GABARAP deletions. (G) Summary of interactions between GABARAP and G3BP1. (H) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged NUFIP2. (I) Quantification of Fig. S4 H. Data (% binding) represents the percentage of the corresponding protein relative to its input. (J) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-G3BP1. (K) GST pull-down assay of in vitro translated and radiolabeled GFP-tagged NUFIP2 with GST or GST-tagged G3BP1 deletions. (L) Immunoblot analysis of interaction between NUFIP2 and G3BP1 in HEK293T cells transfected with FLAG or FLAG-NUFIP2 with 2 mM LLOMe treatment for 30 min. (M) HCM images corresponding to Fig. 8 C. White masks, algorithm-defined cell boundaries. Yellow masks, computer-identified overlap of mTOR and LAMP2. Ctrl, control (untreated cells). AR, autoradiography. Data, means ± SEM; HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). **, P < 0.01, ANOVA. See also Figs. 7 and 8. Source data are available for this figure: SourceData FS4.

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