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. 2014 Oct 30;13(18):2944–2951. doi: 10.4161/15384101.2014.947197

Figure 1.

Figure 1.

ECT2 BRCT is a PAR binding domain. (A) Schematic structure of ECT2, containing BRCT repeats, a DH-type RhoGEF domain, a pleckstrin homology (PH) domain and a polybasic cluster (PBC). (B) The interaction between PAR and GST (negative control), GST-CHFR (positive control), or GST-ECT2 BRCTs was examined by GST pull-down and dot blot with anti-PAR antibody. PAR was blotted and shown as the input. (C) The interaction between biotin-PAR and the recombinant proteins in (A) was examined by the reciprocal pull-down with streptavidin beads and blotted with anti-GST antibody. Recombinant proteins were blotted and shown as the input. (D) The affinity between PAR and GST-ECT2 BRCTs was measured by ITC (left); the affinity between ADP-ribose and GST-ECT2 BRCTs was measured by ITC (right). Titration of PAR or ADP-ribose was injected into a solution containing the purified protein. The inset shows the fit of the data to an equilibrium binding isotherm. The fit provides an equilibrium dissociation constant (KD) for the binding of PAR or ADP-ribose to the protein.