Isolation of CERS2-deficient clones. (A) The target site of the TALEN-CERS2 pair in the human CERS2 gene and indel analysis in TALEN-CERS2–treated (TAL-CERS2) clones. The target sequences (CERS2 Fw and Rv) are located in intron 1 and exon 2, respectively. The sequence between the TALEN target sequences contains the start codon (yellow box) and the splicing acceptor (SA) at the end of intron 1 (blue lined box). Green boxes highlight the portions corresponding to the start codon and the SA. Deletions are shown in red and their lengths are specified on the right of the sequences; (B) Western blot analysis of CERS2 in the TAL-CERS2 clones. Cell lysates from the parent cells and CERS2-deficient mutants were immunoblotted with anti-CERS2 antibody (top) and anti-α-tubulin antibody (middle). Part of the enlarged view of the top image is shown at the bottom. The arrow and arrowhead indicate CERS2; P, parent cell line; 16, TAL-CERS2#16 clone; 18, TAL-CERS2#18 clone; 13, TAL-CERS2#13 clone; (-), no transfection; M, mock; and C2, CERS2 cDNA restoration. Note that overexpression of CERS2 resulted in formation of high molecular weight aggregates.