Ku86 Regulates Pathway Choice in Human Cells Figure 7. Independent confirmation of microhomology-mediated end joining in C-NHEJ mutant cell lines. (A) Reporter substrate biased for use by microhomology-directed NHEJ (A-NHEJ). The reporter has been designed such that cleavage with Eco47III and EcoRV results in a blunt- ended linear substrate with 6-bp direct repeats (boxes) at both ends. C-NHEJ joining will result in the retention of some of both repeats whereas A- NHEJ should generate a single repeat, which is a substrate for BstXI. This figure is excerpted from Verkaik et al., 2002, Eur. J. Immunol., 32:701. (B) The experimental scheme for analysis of the plasmids recovered from transfected cells. The plasmids were subjected to PCR using one radiolabeled (asterisk) primer. The PCR products were then subjected to BstXI restriction enzyme digestion. (C–F) Left Panels: Autoradiograms of representative microhomology assays using the indicated cell lines. The size of the primary PCR product (180 bp) and the BstXI cleavage product (120 bp) are indicated. Right Panels: Three independent experiments similar to the ones shown on the left were quantitated with a phosphoimager and averaged. doi:10.1371/journal.pgen.1000855.g007 PLoS Genetics | www.plosgenetics.org 8 February 2010 | Volume 6 | Issue 2 | e1000855