HAP1 cells edited at each ARF locus do not present defects in morphology or coats recruitment. ARFs were tagged at their endogenous locus with the self-labeling enzyme Halo in a HAP1 haploid cell line. (A) Live cells were stained with the Halo substrate JFX650-CA. (B) Immunoblot to detect ARF-Halo fusion proteins in HAP1 KI lysate with anti-Halo antibodies and anti-βActin primary antibody as a loading control. (C and D) HAP1 WT and ARF4^EN-Halo KI cells were immunostained with anti-KDELR and secondary antibodies conjugated to Alexa488 (C). Quantification of the normalized mean fluorescence intensity of KDELR at the Golgi area measured in HAP1 WT and ARF4^EN-Halo KI cells as described in Materials and methods. Scatter dot plot shows the mean values (black dots) and SEM for each biological replicate (n = 3). All data points (individual cells) are shown as gray dots (n ≥ 10 cells per replicate). Unpaired, two-tailored t test (ns = non-significant, P > 0.05; D). (E–G) HAP1 WT and ARF^EN-Halo KI cells were immunostained with anti-COPI and anti-clathrin and secondary antibodies conjugated to Alexa488 (E). Quantification of the mean fluorescence intensity of COPI (F) and clathrin (G) at the Golgi area measured in HAP1 WT and ARF^EN-Halo KI cells as described in Materials and methods. Scatter dot plots show the mean values (black dots) and SEM for each biological replicate (n ≥ 3). All data points (individual cells) are shown as gray dots (n ≥ 10 cells per replicate). Ordinary one-way ANOVA versus WT (ns, P > 0.05). (H and I) ARF1^EN-SNAP+ARF4^EN-Halo (H, magenta/green) and ARF4^EN-SNAP+ARF5^EN-Halo (I, magenta/green) double KI HAP1 cells were labeled with JF552-CA and JFX650-BG. Norm. Mean Fluo. Int. Golgi = normalized mean fluorescence intensity at Golgi. All images were background subtracted and smoothed with a Gaussian filter as described in Materials and methods. Scale bars are 10 µm. Source data are available for this figure: SourceData FS2.