STED microscopy of double-edited ARF1-ARF4 and ARF4-ARF5 cells suggests distinct functions. (A and B) ARF1^EN-2xV5/ARF4^EN-2xALFA (A, green/magenta) and ARF4^EN-2xV5/ARF5^EN-2xALFA (B, green/magenta) double KI HeLa cells were fixed and immunostained with anti-ALFA and anti-V5 primary antibodies and secondary antibodies labeled with either ATTO647N or AlexaFluor594 to be able to perform dual-color STED experiments. (C) ARF1^EN-2xALFA KI HeLa cells were fixed and immunostained with a single anti-ALFA and secondary antibodies labeled with ATTO647N (magenta) and AlexaFluor594 (green). Line profiles in each panel correspond to the dotted boxes in the cropped images. 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 (A and B) or 10 and 2 µm in the cropped images (C).