ARF1 and ARF3 are the sole TGN-localized ARF members. (A–E) ARF1^EN-, ARF3^EN-, ARF4^EN-, and ARF5^EN-2xALFA KI HeLa cells were immunostained with anti-ALFA tag (magenta) and anti-Golgin97 (green) and secondary antibodies labeled with either ATTO647N or AlexaFluor594 to be able to perform dual-color STED experiments. (E) ARF1^EN-, ARF3^EN-, ARF4^EN-, and ARF5^EN-2xALFA KI HeLa cells were treated with nocodazole (33 µM) for 3 h and immunostained as described above. (F) Scatter dot plot with mean and SD represents the quantification of the distances from the edge of the ARF-labeled cisternae and Golgin97-labeled TGN cisternae measured in nocodazole-treated fixed cells as described in Materials and methods. ARF1 n = 10; ARF3 n = 12; ARF4 n = 10; ARF5 n = 12; GM130 n = 17. n = number of ministacks from at least three independent experiments. Images were deconvolved, background subtracted (A–D), and smoothed with a Gaussian filter (A–E) as described in Materials and methods. Scale bars are 5 and 1 µm in the cropped images.