Isolation of UGCG-deficient and CERT/UGCG double-deficient clones.

A, Target sites of TALEN-UGCG pair (TAL-ModA-UGCG) in human UGCG gene. The sequences are located in exon 6, which contains the codon of the 195th arginine (R195) essential for the activity. The target sites are shown in bold and the codon of R195 is shown in red. The numbers on the right and left of the sequence indicate the sequence numbers from the A of the translation initiation codon, based on UGCG mRNA (accession number D50840). B, Surface expression of StxRs on TALEN-UGCG–treated HeLa cells. HeLa cells were treated with TALEN-UGCG (colored histogram with black line) or empty vectors (blue line), and the cells were stained with Alexa-555-Stx1B. C, Surface expression of StxRs on TAL-UGCG clones (TAL-UG#7 and #3) and a TAL-CERT/UGCG clone (TAL-CE#14UG#2). The clones were stained with Alexa-555-Stx1 B (colored histogram with black line) or not (magenta line) and HeLa-mCAT#8 cells were stained with Alexa-555-Stx1 B (blue line). D, Indel analysis of UGCG gene in TAL-UGCG (TAL-UG#7 and #3) and TAL-CE#14UG#2 clones. Deletions are shown in red and their lengths are specified on the right of the sequences. The predicted proteins are indicated based on the recommended description (see Materials and Methods) [50]. E, Metabolic labeling of lipids with radioactive galactose. TAL-UG#7, -UG#3 and -CE#14UG#2 cells were labeled with [14C]galactose for 16 h, and lipids extracted from the cells were separated by HPTLC. Radioactive image of an analyzed TLC plate is shown. MGDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacylglycerol (Galα1-4GalDG); PC, phosphatidylcholine.