FIGURE 4.

The LKEX₄EV motif does not affect the interaction between CtIP with BRCA1 and Nbs1. A, CtIP dimerization occurs regardless of DNA damage. 293T cells were co-transfected with HA-CtIP and Myc-CtIP. The cells were treated with IR radiation (20 Gy) and allowed to recover for 1 h. After recovery, the cells were collected and lysed. Anti-HA IP was performed, followed by anti-Myc Western blotting (left panel). 293T cells were co-transfected with Myc-CtIP 1–897 and HA-CtIP 1–897 or ATM targeted sites mutant (S664A/S745A). The cells were treated with IR radiation (20 Gy) and allowed to recover for 1 h. After recovery, the cells were collected and lysed. Anti-Myc IP was performed, followed by anti-HA Western blotting (right panel). B, 293T cells were co-transfected with Myc-BRCA1 and HA-tagged CtIP 1–897 or indicated mutants. Anti-HA IP was performed, followed by anti-Myc Western blotting. C, SF21 insect cells were co-infected with baculoviruses expressing FLAG-Nbs1 and the indicated GST-tagged CtIP constructs. GST pull-down experiments were carried out using GST beads, and immunoblotting was performed using anti-FLAG M2 antibody. IP, immunoprecipitation.