Fig 6. Analysis of DNA end joining in Cip29-depleted egg extract.
(A) Sequencing gel analysis of DNA end joining in X. laevis egg extracts using a defined repair substrate. Linearized repair substrate was incubated at 1ng/μl in mock-depleted, Mre11-depleted or Cip29-depleted extract (21°C, 6h). Where indicated, DMSO was added to a final concentration of 0.4% and DNA-PKi (NU7441), dissolved in DMSO, was added to a final concentration of 8μM. Cip29-depletion was performed with two different α-Cip29 antibodies (ΔCip29-1 and ΔCip29-2). Following incubation, plasmid DNA was recovered, digested with TaqαI and BstXI, resolved on a 20% denaturing polyacrylamide gel and exposed to a phosphorimager screen. Linearized substrate added to mock-depleted extract and processed immediately serves as an unrepaired control (0h). (B) Western blot of immunodepleted extracts from (A) indicating the depletion efficiency for Cip29 with both α-Cip29 antibodies. Uhrf1 serves as a loading control. Molecular weight markers (kDa) are indicated on the right.