Fig. 6. Endogenous XRCC1 stimulates AP endonuclease activity in whole-cell extracts. (A) Reactions were carried out on an oligonucleotide harboring a single THF site as described in Materials and methods with increasing amounts of total protein as indicated. The products of the reactions were separated by denaturing 20% PAGE. The positions in the gel of the substrate and the product of the reaction are indicated in the figure. XRCC1-deficient (EM9) or XRCC1-proficient (EM9-X) cell lines were used to prepare the extracts. Where indicated, standard reactions were supplemented with 1.4 pmol of purified human XRCC1 (+XRCC1). (B) Plot of the percentage of DNA substrate cleaved by the AP endonuclease activities versus total amount of protein used in the reaction. Each point represents the average and standard deviation from three independent experiments. (C) XRCC1 and APE1 contents were determined by western blot analysis on 30 µg of each protein extract.