Type I ARFs define tubular-vesicular clathrin-positive structures on the TGN and the cell periphery. (A–D) ARF1^EN-, ARF3^EN-, ARF4^EN-, and ARF5^EN-Halo (magenta) and SNAP-CLCa^EN (green) double KI HeLa cells were labeled with JF571-CA together with JFX650-BG (A and C) or JFX650-CA and JF585-BG (B and D) and imaged with a STED microscope. ARFs tubular-vesicular structures are highlighted by white arrows and clathrin clusters by yellow arrows. (E) Double KI cell lines were labeled with JFX650-CA and JF585-BG and then treated with nocodazole (33 µM) for 3 h. (F) Scatter dot plot with mean and SD represents the quantification of the distances from the edge of the ARF-labeled cisternae to the center of clathrin vesicles measured in nocodazole-treated live cells as described in Materials and methods. ARF1 n = 33; ARF3 n = 30; ARF4 n = 19; ARF5 n = 19. n = clathrin vesicles from at least three independent experiments. All images were deconvolved, background subtracted, and smoothed with a Gaussian filter as described in Materials and methods. Scale bars are 5 and 1 µm in the cropped images.