FIGURE 4.

Sumoylation of free and DNA-bound TDG in the presence and absence of HeLa nuclear extract. A–C, immunoblot analysis of TDG modification in the presence and absence of nuclear extract for free TDG (A), TDG bound to nonspecific DNA (B), and TDG bound to AP-DNA (C). Reactions were performed in sumoylation buffer A with 0.1 μm E1, 0.5 μm E2, 0.8 μm TDG, and 5 μm SUMO-1 and were initiated by the addition of 10 mm ATP. As indicated, some reactions contained 15 μg of HeLa NE and/or 5 μm S2A (a SENP inhibitor). Samples were extracted from the reaction at the indicated time points, diluted 10-fold into buffer A, and quenched with 5×sample buffer before separation via SDS-PAGE under reducing conditions and transfer to nitrocellulose membranes. TDG was detected via immunoblotting with anti-His antibody and a fluorescent secondary antibody.