ARFs segregate on different early secretory membranes. (A–I) ARF1^EN-, ARF4^EN-, and ARF5^EN-2xALFA KI HeLa cells were immunostained with anti-ALFA (magenta) and either anti-ERGIC53 (A–C, green) or anti-GM130 (D–F, green) and secondary antibodies labeled with either ATTO647N or AlexaFluor594 to be able to perform dual-color STED experiments. Line profiles in each panel correspond to the dotted boxes in the cropped images. (G–I) ARF1^EN-, ARF4^EN-, and ARF5^EN-2xALFA KI cells were treated with nocodazole (33 µM) for 3 h and immunostained as described above. (J) Scatter dot plot with mean and SD represents the quantification of the distances from the edge of the ARF-labeled cisternae and cis-Golgi cisternae measured in the nocodazole-treated fixed cells as described in Materials and methods. ARF1 n = 9; ARF3 n = 11; ARF4 n = 15; ARF5 n = 14; Golgin97 n = 17. n = number of ministacks from at least three independent experiments. (K) Graphical summary of the nanoscale localization of ARFs on early secretory membranes. Images were deconvolved, background subtracted (A–F), and smoothed with a Gaussian filter (A–I) as described in Materials and methods. Scale bars are 5 and 1 µm in the cropped images.