SOD1 is transported to lysosomes. (A–C) Confocal images of N2a cells expressing mCherry-GFP-SOD1(A4V) (A) or mCherry-GFP-SOD1(G85R) (B) under control (Ctrl) or starvation (Strv) conditions. Quantification of mCherry-only puncta is presented as mean ± SEM (A4V Ctrl, n = 20; A4V Strv, n = 20; G85R Ctrl, n = 20; G85R Strv, n = 20) (C). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (D and E) Confocal images of COS7 cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (D). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 19) (E). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (F and G) Confocal images of 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl), starvation (Strv), or starvation plus Baf (Strv + Baf) conditions (F). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 28; Strv + Baf, n = 23) (G). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (H) Immunoblotting shows the levels of TMEM192-3×HA, CTSL (lysosomal protein), EMC1 (ER protein), TOMM20 (mitochondrial protein), and GAPDH (cytosolic protein) in total cell lysates and isolated lysosomes. Lyso, isolated lysosomes; CTSL, cathepsin L.