Fig. 1: Determination of the role of Poly-D/E regions on BCOR binding to KDM2B.

A Schematic representation of the domain architecture of BCOR. Three Poly-D/E regions on linker are highlighted with light red for Poly-D/E^1st, red for Poly-D/E^2nd, and dark red for Poly-D/E^3rd. B Truncations of BCOR used in this study. C Binding affinity between BCOR^ANK-linker and KDM2B^F-box-LRRs/SKP1 was measured by ITC with buffer containing 50 mM or 150 mM NaCl. D Binding affinity measurement between dimer of PCGF1^RAWUL/indicated BCOR truncation and the KDM2B^F-box-LRRs/SKP1 dimer using ITC with buffer containing 150 mM NaCl. N/D: Cannot be determined. For (C, D), The K_d values are shown as mean ± SD for triplicate experiments. E The importance of Poly-D/E regions on the linker of BCOR binding to KDM2B/SKP1 is assessed using Co-IP assay. Expressing plasmids for BCOR (wild or mutant), PCGF1, KDM2B or SKP1, were co-transfected into HEK293T cells. Co-IP was performed with anti-Flag magnetic beads, after 48 h transfection. The western-blotting data is representative of three independent experiments. F Relative level of KDM2B-HA or PCGF1-strep protein co-purified with BCOR-Flag as shown in (E). Data are represented as mean ± SD for triplicate experiments.