Research Paper Volume 10, Issue 6 pp 1454—1473

Figure 3. The NER excision activity of NMR and mouse cell extracts. (A) Representative phospho-image of product analysis for mouse (lanes 2-6) and NMR (lanes 7-11) cell extracts. The substrate DNA was incubated for 45 min at 30°C with cell extracts (15 nM substrate DNA, 0.3 mg/ml extract proteins). The excision products were detected by annealing to a specific template containing 5ʹ-GpGpGpGpG overhang, which was then end-labeled using α-[³²P]-dCTP and Taq DNA polymerase. The reaction products were resolved on a 10% denaturing polyacrylamide gel. [³²P]-5ʹ end-labelled oligonucleotides were used as length markers (lane M). nFlu-DNA without cell extract was used as a negative control (lane 1). (B) Quantification of the levels of excision products based on three independent experiments. The activity in the extract of mouse non-irradiated cells was taken as 1. The data are the mean ±SD, n=3. Grey and black lines correspond to mouse and NMR cell extracts, respectively.