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. 2017 Jun 15;28(12):1676–1687. doi: 10.1091/mbc.E16-12-0863

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© 2017 Bottanelli et al. 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 1: — Endogenous tagging of ARF1 highlights Golgi-derived tubular structures. Cells either transiently overexpressing ARF1-Halo (a) or expressing gene-edited ARF1^EN-Halo (b) were labeled with SiR-CA and imaged with a confocal microscope. (b) A Golgi-derived tubule is highlighted by arrows. (c) Total numbers of tubules/cell and Golgi-derived tubules/cell were quantified in both cells both live and after fixation with 4% PFA. Result of a two-tailed, unpaired t test. ***p < 0.001, ****p < 0.0001 (n = 10 cells). (d) Gene editing was validated via Western blot using an antibody that recognizes class I ARFs (ARF1 and ARF3) due to the high protein sequence homology. The added amounts of ARF1^EN-Halo (∼35%) and unedited ARF (∼70%) in the ARF1^EN-Halo cell line match the amount of ARF1 (set to 100%) in CCL-2 HeLa cells. (e, f) ARF1^EN-Halo cells were imaged on a custom-built STED setup. (g) The average width (FWHM) of the Golgi tubules was 110 ± 21 nm (n = 20). All STED images were deconvolved; the line profile represents raw image data. All error bars represent SD. Scale bars, 10 μm (a, b), 5 μm (cropped images, a, b), 5 μm (e), 2 μm (f).