Fig. 2

ACBD3 contributes significantly to Golgi localization of KDELR at steady state. A–D Confocal micrographs of HeLa cells expressing KDELR1-mCherry (A), KDELR2-mCherry (B), and KDELR3A-mCherry (C), showing that depletion of ACBD3 results in significant re-distribution of all three isoforms of KDELRs from the Golgi to the ER in vivo, which could be restored by exogenous expression of RNAi-resistant EGFP-ACBD3. For quantification (D), the percentage of fluorescent intensity of Golgi-localized KDELRs over the total KDELR-mCherry were quantified and plotted onto the histogram as average ratio with s.d (n = 20 ~ 25) (ACBD3-KD vs Control, **p < 0.001; ACBD3-KD/rescued vs ACBD3-KD, ^##p < 0.01; ^###p < 0.001). E Schematic illustration of CRISPR-Cas9-mediated insertion of 3xFlag-mCherry tagging at the C-terminal end of endogenous KDELR1. F Confocal micrographs of KDELR1^endo-3xFlag-mCherry in HeLa cells showing that depletion of ACBD3 results in re-distribution of KDELR1^endo-3xFlag to the ER in vivo. G Endogenously tagged KDELR1-3xFlag-mCherry pulls down ACBD3 and ArfGAP1/3, respectively. HeLa cells with KDELR1-3xFlag-mCherry was lysed and subjected to immunoprecipitation with anti-RFP antibody, followed by western blot analysis. H Confocal results showing that hGH-GFP-KDEL expression induces endogenously 3xFLAG-mCherry-tagged KDEL receptor to re-distribute to the ER in HeLa cells. The bar graph to the right summarizes the quantification of these results. (***p < 0.001) Scale bars = 10 μm