Figure 4. CtIP 5′-flap endonuclease activity shows epistasis with MRE11 3′–5′ exonuclease activity in counteracting Ku binding to seDSBs.
(a) Schematic of CtIP domains with the position of N289 and H290 residues. (b) Immunoblotting of extracts from U2OS T-REx cells stably transfected with control or siRNA-resistant wild-type (WT) or NAHA HA-CtIP-expressing plasmids, and transfected with the indicated siRNA. (c) Quantification of Ku focus number in replicating cells transfected as in b and treated with CPT. (d) Immunoblotting of extracts from U2OS T-REx cells stably transfected with control or siRNA-resistant WT HA-CtIP and HA-MRE11 or NAHA HA-CtIP and H63N HA-MRE11, and transfected with the indicated siRNAs. (e) Quantification of Ku foci in U2OS T-REx stably transfected with control or the specified siRNA-resistant constructs and transfected with the indicated siRNAs. Error bars are s.d. Significant differences between specified pairs of conditions, as judged by t-test, are highlighted by stars (*P<0.05; **P<0.01; ***P<0.0005; ****P<0.0001). NS, non-significant difference.