ARF1 and ARF4 define different populations of ERGICs. (A and B) ARF1^EN-Halo and ARF4^EN-Halo KI HeLa cells were transfected with a plasmid encoding for SNAP-ERGIC53. Live cells were stained with the Halo substrate JF571-CA and the SNAP substrate JFX650-BG and imaged with a STED microscope. (A ⅰ–ⅲ and B ⅰ–ⅲ) White arrows indicate Golgi-associated ERGICs devoid of ARF1 (A i and ii) and positive for ARF4 (B i and ii) and peripheral ERGICs positive for either ARF1 (A iii) or ARF4 (B iii). (C) ARF1^EN-SNAP and ARF4^EN-Halo double KI HeLa cells were labeled with JF571-CA and JFX650-BG and imaged with a STED microscope. (C ⅰ–ⅲ) Overview of Golgi area showing ARF4 Golgi-associated ERGICs devoid of ARF1 (C i, white arrow) and ARF1/ARF4 segregated tubular-vesicular structures (C ii and iii, yellow and white arrows, respectively). (D) Distribution of peripheral ERGIC populations defined by ARF1 and ARF4. The percentage of peripheral ERGICs positive for either ARF1 or ARF4 was calculated in images of either ARF1^EN or ARF4^EN-Halo KI cells transfected with a plasmid encoding for SNAP-ERGIC53. The percentage of ARF1- and ARF4-positive peripheral ERGICs was calculated in images of ARF1^EN-SNAP and ARF4^EN-Halo double KI cells transfected with a plasmid encoding for YFP-ERGIC53. ARF1+ARF4/ERGIC n = 13; ARF1/ERGIC n = 10; ARF4/ERGIC n = 10. n = number of cells from at least three independent experiments. Error bars represent mean and SD. (E) Gallery of peripheral ERGICs showing ARF1/ARF4 segregated nanodomains. (F) Graphical summary of the nanoscale localization of ARF1 and ARF4 on ERGICs. All images were deconvolved, background subtracted, and smoothed as described in Materials and methods. The brightness in the crops was enhanced to highlight the dim peripheral structures. Scale bars are 5 and 1 µm in the cropped images.