Figure 6.

Human cells harboring the Nbs1-2A mutant are defective in the checkpoint response to IR. (A) Sequence alignment of Xenopus and human Nbs1 around critical residues in the first BRCT domain. (B) HeLa cells were infected with lentiviruses expressing GFP (lanes 1–4) or either wild-type or 2A versions of human Nbs1 (lanes 5 and 6). Control or Nbs1 siRNA was transfected into these infected cells as indicated. Cells were mock-treated or exposed to IR. Cell extracts were prepared 2 h later and processed for immunoblotting with the indicated antibodies. (C) HeLa cells were transfected control vector (lanes 1 and 2) or a vector expressing either wild-type (lanes 3 and 4) or 2A versions (lanes 5 and 6) of FLAG-tagged human Nbs1. At 48 h after transfection, cells were treated as described in B. Anti-FLAG immunoprecipitates from cell lysates were immunoblotted with anti-TopBP1 and anti-FLAG antibodies.