Figure 3.

Effect of gene disruption on human gpERAD. (A) Strategy for TALEN-mediated gene disruption. Cleavage of the exon 1 encoding the signal sequence of human EDEM1, EDEM2, or EDEM3 with the designed TALEN facilitates subsequent homologous recombination with the respective targeting vector containing both positive and negative selection markers. (B) Results of screening. The presence (amp +) or absence (amp −) of the ampicillin-resistance gene in targeted alleles was checked by genomic PCR. (C) Genomic PCR to confirm homologous recombination in HCT116 cells of various genotypes. Asterisks denote nonspecific bands. (D) RT-PCR to amplify cDNA corresponding to hEDEM1, hEDEM2, or hEDEM3 mRNA expressed in HCT116 cells of various genotypes. The full-length cDNA and cDNA lacking the exon 6 were amplified as hEDEM1. (E) Doubling time of WT and three hEDEM-KO cells (n = 4). (F) Display, similarly to Fig. 2 C, of oligosaccharides whose contents in kifunensine (Kif; 10 µg/ml, 12 h)-treated WT HCT116 and three hEDEM-KO cells exceeded those in kifunensine-untreated WT cells with an increase over WT (%), as determined from isomer composition of their N-glycans (Fig. S3, F and G), which was completed once. Asterisks denote oligosaccharides whose contents did not exceed those in kifunensine-untreated WT cells. (G) Immunoblotting of cell lysates prepared from kifunensine-untreated or -treated WT HCT116 cells and from three hEDEM-KO cells using anti-human ATF6α (hATF6α) antibody. (H) Immunoblotting of cell lysates prepared from WT and hEDEM2-KO HCT116 cells with or without endoglycosidase H treatment. hATF6α* denotes the nonglycosylated hATF6α.