Gene editing with CRISPR-Cas9 highlights the endogenous localization of ARF GTPases. (A–H) ARFs were tagged at their endogenous locus with the self-labeling enzyme Halo (A–D) or 2xALFA tag (E–H). An additional LAP tag linker was added in the case of ARF3 (B). Live cells were stained with the Halo substrate JF646-CA (A, C, and D) and JFX650-CA (B). Fixed cells were immunolabeled with an anti-ALFA primary antibody and ATTO647N-conjugated secondaries. (I) Scatter dot plot with mean and SD represents the quantification of the mean fluorescence intensity at the Golgi area measured in the ARF^EN-2xALFA KI cells as described in Materials and methods. ARF1 n = 22; ARF3 n = 22; ARF4 n = 24; ARF5 n = 20; n = number of total cells from at least three independent experiments. Yellow arrows highlight structures positive for endogenoulsy tagged ARFs. All images were smoothed with a Gaussian filter and background subtracted as described in Materials and methods. Mean fluo. int. Golgi area (A.U.) = mean fluorescence intensities at the Golgi area in arbitrary units. Scale bars are 10 and 2 µm in the cropped images.