MANI-FM localizes to a cis/medial position in the Golgi stack. HeLa cells expressing MANI-FM were cultured in the presence of AP, and then fixed and processed for cryoimmunolabeling with an anti-HA antibody (10-nm gold particles). (a) Localization of MANI-FM in the cis-/medial cisternae, with a clathrin bud (arrow) marking the trans side. (b) An example of the preferential rim distribution of MANI-FM. (c) An enlargement of b, with MANI-FM in a peri-Golgi carrier (arrowhead). (d–f) Quantification of the distribution of MANI-FM across the stack (d), in the central versus the rim sections of the cisternae (e), and in peri-Golgi vesicles and tubules (f). Data are mean ± SEM, obtained from analyses of 28 stack profiles. (g) Localization of MANI-FM in a Golgi stack under confocal microscopy. MANI-FM (green) localizes between GM130 (blue) and TGN46 (red). The white arrow across the stack was used for line-scan analyses. The image is representative of >30 stacks analyzed. (h) Fluorescence intensity distribution of markers along the line scan (arrow in g). The fluorescence intensities were normalized to their respective peak values. The image and the corresponding quantitation are representative of at least 30 Golgi ministacks from three independent experiments. For this experiment, n = 30. Bars: (a) 270 nm; (b) 240 nm; (c) 60 nm; (g) 1 µm.