CAX localizes to acidic organelles. (a and b) Subcellular distribution of CAX in neural crest cells. (a) Confocal (top) and transmitted light (bottom) micrographs of exemplar neural crest cells expressing GFP-CAX. Inset shows a magnified image highlighting large (open triangles) and small (closed triangles) GFP-positive structures. (b) Images of GFP-CAX–expressing cells labeled with Lysotracker red (Lyso.). (Left) Images of large vesicles from cells loaded with 30 nM Lysotracker. (Right) Magnified images of small vesicles from independent cells loaded with 300 nM Lysotracker. Intensity plots of red and green fluorescence between the white lines in the overlaid image are shown below the images. (c–f) Subcellular distribution of CAX in human cells. (c) Confocal micrographs of the indicated cell type transiently transfected with GFP-CAX and fixed before imaging. (d) Distribution of GFP-CAX in live HeLa cells labeled with 100 nM Lysotracker red. (e) Distribution of GFP-CAX in fixed HeLa cells coexpressing LAMP1-mRFP. (f) Immunocytochemistry analysis using an antibody raised to LAMP1 in SHSY5Y cells or fibroblasts expressing GFP-CAX. Bars: (a and b) 5 µm; (c–f) 10 µm.