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. 2014 Oct 27;33(23):2860–2879. doi: 10.15252/embj.201489017

Figure 5. CtIP interacts with Cdh1 through a conserved KEN box.

Figure 5

  1. Schematic representation of human CtIP protein with its coil-coiled domain (45–160), Sae2-like domain (790–897), and putative KEN boxes (179–181 and 467–469). Conservation of KEN box at 467 is shown for the indicated species.
  2. HEK293T cells were transfected with HA-Cdh1 in combination with indicated FLAG-CtIP plasmids. Cells lysates were used for anti-HA immunoprecipitations. Western blotting was performed with the indicated antibodies for whole-cell lysate or immunoprecipitations.
  3. HeLa nuclear extract (NE) was incubated with GST fusion proteins with the indicated full-length (1–897) CtIP variants or CtIP fragment (166–487) variants. GST pull-downs were immunoblotted for Mre11 and Cdh1.
  4. HEK293 Flp-In T-REx cells were induced to express GFP-CtIP-wt using doxycycline and were transfected with His-tagged ubiquitin (“His-Ub”) along with control siRNA or Cdh1 siRNA. Before lysis, cells were treated with proteasome inhibitor MG-132 for 4 h. Cell lysates were used for Ni-NTA precipitations. Total cell lysates (“input”) and Ni-NTA pull-downs (“PD”) were immunoblotted for the indicated proteins.
  5. HEK293 Flp-In T-REx cells were induced to express GFP-CtIP-wt or GFP-CtIP-K467A and transfected with His-tagged ubiquitin (“His-Ub”), and treated with proteasome inhibitor MG-132 for 4 h. Cell lysates were subsequently used for Ni-NTA pull-down. Total cell lysates (“input”) and Ni-NTA pull-downs (“PD”) were immunoblotted for the indicated proteins.
  6. U2OS Flp-In T-REx cells were induced to express GFP-CtIP-wt or GFP-CtIP-K467A and were transfected with control siRNA or siCtIP. Cell lysates were processed for immunoblotting for CtIP and actin (upper right panel). U2OS cells were then imaged every 5 min for GFP expression or phase contrast using live-cell microscopy. Representative stills from live-cell imaging are shown, in which anaphase onset was used as a reference time point (left panels). Quantifications of the average nuclear GFP signal from time-lapse movies are indicated for GFP-CtIP-wt (n = 12) and GFP-CtIP-K467A (n = 18) (lower right panel).