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. 2015 Mar 17;10(3):e0119869. doi: 10.1371/journal.pone.0119869

Fig 2. Establishment of claudin-2 knockout clones in MDCK II cells.

Fig 2

(A) Immunofluorescence analysis of claudin-2 and ZO-1 in control MDCK II cells (CTL) and claudin-2 knockout clones (KO 1–5). Claudin-2 staining at cell-cell contacts was completely lost in claudin-2 knockout clones. Scale bar = 10 μm. (B) Immunoblots of claudin-2 and E-cadherin in control MDCK II cells and claudin-2 knockout clones. A claudin-2 band of ~22 kDa was absent in claudin-2 knockout clones, but faint bands of a lower molecular weight than wild-type claudin-2 band were observed. (C) DNA sequences of the TALEN targeting site in each allele of claudin-2 knockout clones. One type of mutation was found in the alleles of claudin-2 knockout clone 1 (KO 1), two types in the alleles of clones 3 and 5 (KO 3 and 5), three types in the alleles of clone 2 (KO 2), and four types in the alleles of clone 4. Dashes indicate loss of nucleotides, green letters indicate additional nucleotides, and a yellow letter indicates an altered nucleotide. Loss of initiating codon or frame shift was confirmed for all alleles. (D) Genomic PCR analysis of control and claudin-2 knockout clones using primers for TALENs and claudin-2 DNAs. A clone stably expressing TALEN was used as a positive control (PC). None of the PCR products for TALENs was detected in claudin-2 knockout clones.