Figure 1.
XtopBP1 interacts in a regulated manner with the Xnbs1 subunit of the MRN complex in Xenopus egg extracts. (A) Control and anti-XtopBP1 immunoprecipitates (IP) from egg extracts containing pA-pT were immunoblotted for Xnbs1. (B) Control and anti-Xnbs1 immunoprecipitates from egg extracts containing pA-pT were immunoblotted for XtopBP1. (C) Anti-FLAG antibody beads containing no recombinant protein (lanes 1 and 2), Xmre11-FLAG (lanes 3 and 4), Xnbs1-FLAG (lanes 5 and 6), and Xrad50-FLAG (lanes 7 and 8) were incubated in egg extracts lacking or containing pA-pT. Beads were retrieved and immunoblotted with anti-XtopBP1, anti-Xatm, and anti-FLAG antibodies. (D) Anti-FLAG antibody beads containing no recombinant protein (lanes 3 and 4) or HF-XtopBP1 (lanes 5 and 6) were incubated in egg extracts containing 35S-labeled Xnbs1 with or without pA-pT. The beads were reisolated, and bound 35S-labeled Xnbs1 was detected by SDS-PAGE and phosphorimaging. Lanes 1 and 2, aliquots of initial extracts lacking or containing pA-pT. (E) Anti-FLAG antibody beads containing no recombinant protein (lanes 1 and 2), Xnbs1-FLAG (lanes 3 and 4), MRN complex (Xmre11, Xrad50-FLAG, and Xnbs1) (lanes 5 and 6), or MR complex (Xmre11 and Xrad50-FLAG) (lanes 7 and 8) were incubated in egg extracts lacking or containing pA-pT. Beads were retrieved and immunoblotted with antibodies against XtopBP1 and Xatm.