Figure 5: CCAR2 and CtIP interacts physically in a constitutive manner.

(a) Cells depleted of the indicated genes were analysed with a PLA using CtIP and CCAR2 antibodies. The average and s.d. of the medians of three independent experiments are plotted on the left. A representative image of each condition is shown on the right. The top graph was obtained with cells unchallenged by exogenous damage. The plots on the bottom side were calculated in cells 1 h after irradiation (10 Gy). Scale bar, 7.5 μm. (b) Protein samples from cells stably transfected with either GFP or GFP–CtIP in undamaged conditions or 1 h after ionizing radiation (10 Gy) were used for immunoprecipitation with anti-GFP resin. Immunoprecipitates were resolved in SDS–PAGE and blotted for the indicated antibodies. The asterisk marks an unspecific band that binds to the resin, and the arrow marks the endogenous CtIP protein. (c) Same as b, but with cells expressing GFP or GFP-CCAR2. (d) GST–CtIP was used as bait for pull-down experiments from whole-cell extracts using cells expressing GFP, GFP-CCAR2 full-length or three deletion mutants of CCAR2, as indicated. Western blots with an anti-GFP antibody using inputs (left) and pull-downs (right) are shown. The red arrows label the position of GFP fusions. (e) Bacterial-purified His₆-CCAR2 was pulled down with bacterial-purified GST–CtIP full-length and deletion constructs. Purified GST was used as a control. The red arrows represent the purified CtIP version. A western blot against CCAR2 is shown at the bottom. (f) A schematic representation of all the deletion constructs used in e. Full-length CtIP and the interaction regions with CCAR2 and the MRN complex are represented at the bottom. (g) PLA foci using CtIP and CCAR2 antibodies in cells expressing GFP-MDC1 that were collected 1 h after irradiation (10 Gy). Scale bar, 7.5 μm.