FIGURE 2.

Phosphomimetic substitutions reduce the ability of Ikaros to bind a DNA probe derived from the pericentromeric heterochromatin region. Nuclear extracts were prepared from HEK293T cells expressing wild-type or mutant Ikaros proteins. Phosphomimetic Ikaros mutants contain substitutions with alanine or aspartate at the indicated amino acids. Equal amounts of Ikaros proteins in nuclear extracts were confirmed by Western blot (upper lane). Gel shifts were performed with two extracts concentrations (lanes 1–12, 3×, 6 μg per reaction; lanes 13–20, 1×, 2 μg per reaction). Gel shifts were performed using radiolabeled probes derived from the murine γ satellite A repeat (A) or the murine γ satellite B repeat (B).