Fig. 5.
Direct interaction between Ku70 and XRCC4. (A) Immunoprecipitation from HeLa cell nuclear extracts using XRCC4 antisera. (Upper) The input material (10%). (Lower) A Western blot of the immunoprecipitated material. C, unirradiated cells; IR, cells irradiated with 45 Gy of γ-rays. (B) Schematic representation of the trifunctional cross-linker sulfo-SBED. (C) Cross-linked polypeptides after incubation of sulfo-SBED-labeled Ku70/80 with XRCC4, followed by photoactivation of the cross-linker. UV cross-linking was performed with only Ku70/80 (lane 1), only XRCC4 (lane 2), or Ku70/80 and XRCC4 (lanes 3 and 4). The products in lane 3 were treated with DTT to reverse the cross-link. The sizes of the molecular mass markers are depicted on the left, and the nature of the various products in the gel have been confirmed by mass spectrometry (MALDI-TOF). (D) Products that could be attached to the aryl azide group of sulfo-SBED that had been linked to Ku70/80. Mixtures contained Ku70/80, DNA-PKCS, ligase IV/XRCC4, and DNA. Lane 1 shows the Ku70/80 preparation alone, and lane 2 shows unbound proteins. Lane 4 shows the products that were precipitated by using streptavidin beads after activation of the aryl azide group and treatment with DTT, with lane 3 showing streptavidin-bound products without activation or DTT treatment.