Ragulator abundance and activity on damaged lysosomes is controlled by NUFIP2. (A) Summary of the quantitative changes in relevant proteins of mTORC1 signaling based on DIA LysoIP LC/MS/MS analysis. FC, fold change (see Table S1, Tab 1). (B) Immunoblot analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells treated with 2 mM LLOMe for 30 min. TMEM192-2xFLAG, control. (C ⅰ–iv) Immunoblot analysis of proteins associated with purified lysosomes (LysoIP) from parental Huh7 WT and NUFIP2-knockout Huh7 cells (NUFIP2^KO) treated with 2 mM LLOMe for 30 min (i); quantification (ii–iv), n = 3. (D) HEK293T cells stably expressing FLAG-Metap2 (control) or FLAG-LAMTOR2 transfected with scrambled siRNA (SCR) or NUFIP2 siRNA (NUFIP2^KD) were treated with 2 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of interaction between RagA and LAMTOR2, n = 3. (E) HEK293T cells stably expressing FLAG-Metap2 (control) or FLAG-LAMTOR2 transfected with GFP or GFP-NUFIP2 were treated with 2 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of interaction between RagA and LAMTOR2, n = 3. (F) HEK293T cells expressing FLAG (control) or FLAG-NUFIP2 were treated with 2 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Data, means ± SEM; †, P ≥ 0.05 (not significant); *, P < 0.05; **, P < 0.01, ANOVA. See also Fig. S3. Source data are available for this figure: SourceData F5.