ARF1 and ARF3 define segregated nanodomains on the TGN and localize to TGN-derived tubular-vesicular trafficking intermediates. (A) ARF1^EN-Halo and ARF3^EN-LAP-SNAP double KI HeLa cells were labeled with JF571-CA and JFX650-BG. (A ⅰ–ⅳ) Crops highlight segregation of gene-edited ARF1 and ARF3 on the Golgi (A i and ii) and their localization on TGN-derived (A iii) and peripheral (A iv) tubular-vesicular trafficking intermediates. ARF3 nanodomains are highlighted by white arrows in the crops or a black arrow in the line profile graph (A i and ii). ARF1- and ARF3-positive tubular-vesicular trafficking intermediates are highlighted by white arrows (A iii and iv). (B) Graphical summary of the nanoscale localization of ARF1 and ARF3. Images were deconvolved, background subtracted, and smoothed with a Gaussian filter as described in Materials and methods. The brightness in the crops was enhanced to highlight the dim distal structures. Scale bars are 5 and 1 µm in the cropped images.