Figure 5. Molecular analysis of cell clones.

A) INDEL analysis of MnlI-TALEN and NcoI-TALEN N-Muli treated cells. The left and right arms of the TALEN pair are indicated in green and red, respectively. The restriction enzyme sites are indicated in blue. Deletions are indicated as dashes. The length of the deletions is indicated on the right of each sequence. B) Western blot analysis of the isolated cell clones. Proteins were prepared from the isolated cell clones and analyzed by Western blot using an anti-Ugt1 antibody. Lanes 1 and 2 correspond to protein extracts prepared from WT and mutant livers [13]. Lane 3 corresponds to untreated N-Muli cells. The arrow indicates the band corresponding to Ugt1. β-tubulin was used to normalize for protein loading. C) Ugt1 glucuronidation activity of the isolated cell clones. Ugt1 glucuronidation activity was determined by the UGT-Glo Assay as described in the Materials and Methods section. The activity determination was determined with microsomes prepared from wt and Ugt mutant mouse liver (1 µg), from wt N-Muli cells (10 µg) and from the isolated cell clones (10 µg, Mnl20, Nco20 and Nco25), as the percentage of luminescent substrate consumed. “+cntrl” indicates microsomes provided by the manufacturers (Promega) as positive control of the experiment. The experiment was repeated twice, and the mean of both experiments is shown.