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. 2010 Oct 15;21(20):3540–3551. doi: 10.1091/mbc.E09-12-1053

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 3. — NHE8-HA is localized to the TGN and MVB. (A) HeLa M-cells were stably transfected with NHE8-HA and immunolabeled with anti-HA antibodies or anti-TGN46 antibodies. A series of confocal Z-stacks (Leica optimized) were taken and are displayed as an extended focus view with the merged image in the right panel. Colocalization of TGN46 (magenta) and NHE8-HA (green) appears white in the merged image. Scale bar, 17 μm. (B and C) HeLa M-cells stably expressing NHE8-HA were prepared for immuno-EM and labeled with antibodies to TGN46 (small arrowheads; 10-nm gold) or the HA tag (15-nm gold). (B) TGN46 and NHE8-HA coimmunolabeled vesicles and cisternae of the TGN adjacent to the Golgi stack (G). (C) NHE8-HA immunolabeling could also be identified within MVB (large arrowheads) on ultrathin cryosections. (D) Quantitation of the labeling density of HA associated with the TGN, MVBs, or vesicular/tubular clusters (VTCs) greater than 500 nm from the Golgi stack from three independent immunolabeling experiments ± SEM. Scale bars, 200 nm.