Generation of ΔUNC50 HeLa cells. (A) Schematic of the UNC50 protein with predicted topology. (B) Amino acid sequence of UNC50 in WT and ΔUNC50 cells. Shaded area with asterisks shows alignment between WT and ΔUNC50 cells. seq., sequence; TM, transmembrane domain. (C) RT-PCR analyses to detect expression of UNC50 in WT and ΔUNC50 cells. As described in the RT-PCR section of Materials and methods, primer pairs 1 and 2 were used to amplify sequences upstream or downstream, respectively, of the locus targeted by the CRISPR system. (D) WT or ΔUNC50 cells were cotransfected with CRISPR-sensitive myc-tagged UNC50 and GFP-tagged SLC30A10. 1 d after transfection, cultures were processed for immunofluorescence. The myc tag was detected using a monoclonal antibody. (E) Quantification of fluorescence intensities from D. For each construct, intensity in WT cells was normalized to 100, and intensity in ΔUNC50 cells was expressed relative to WT (n ≥ 15 cells per condition). (F) Immunofluorescence analyses were performed in WT or ΔUNC50 cells cotransfected with indicated constructs as described in D. (G) Quantification of fluorescence intensities from F as described for E (n = 15 cells per condition). (H) WT or ΔUNC50 cells were transfected with myc-tagged CRISPR-sensitive UNC50. 1 d after transfection, cultures were processed for immunofluorescence. A monoclonal antibody was used to detect the myc tag. Polyclonal antibodies were used to detect the endoplasmic reticulum marker calnexin or the Golgi marker giantin. Bars, 25 µm. (I and J) Quantification of the Pearson’s coefficient for colocalization between UNC50 and calnexin or giantin from H (n ≥ 15 cells per group). *, P < 0.05 by t test; error bars show means ± SEM).