STED microscopy reveals ARF3 clusters segregated from ERGIC and cis-Golgi cisternae. (A and B) ARF3^EN-2xALFA (magenta) KI HeLa cells were fixed and immunostained with anti-ALFA tag and either anti-ERGIC53 (A, green) or anti-GM130 (B, green). Secondary antibodies labeled with either ATTO647N or AlexaFluor594 were used to be able to perform dual-color STED experiments. (C) ARF3^EN-2xALFA KI HeLa cells were treated with nocodazole (33 µM) for 3 h and immunostained with the same primary antibodies described above. Secondary antibodies labeled with either ATTO647N or AlexaFluor594 (GM130)/AlexaFluor568 (ERGIC53) were used. Images were deconvolved, background subtracted (A and B), and smoothed with a Gaussian filter (A–C) as described in Materials and methods. Line profiles in each panel correspond to the dotted boxes in the cropped images. Scale bars are 5 and 1 µm in the cropped images.