Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining
Figure 3
DNA end joining with immunodepleted extracts. HeLa WCE was immunodepleted for the various potential NHEJ proteins indicated, and immunodepletion ( 3-fold) of the target protein was confirmed by western blot (data not shown). The individual immunodepleted extracts were then assayed for DNA end-joining activity at C for 2 hrs. (a) Results of DNA end-joining reactions performed in the absence of 5% PEG. (b) Results of DNA end-joining reactions performed in the presence of 5% PEG. All reactions were performed in triplicate and error bars indicate the standard deviation. (c) Wortmannin-insensitive DNA end joining is detectable in the DNA-P immunodepleted HeLa WCE in the absence of PEG. Reactions were run in the absence of 5% PEG and where indicated, in the presence of 10 M wortmannin at C for 2 hrs. (L) T4 DNA ligase positive control; () negative control; (ID) DNA-P-immunodepleted WCE.
