FIGURE 4.

Phosphomimetic substitutions alter the DNA binding affinity of Ikaros toward the upstream regulatory element of the TdT gene. 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×, 6μg per reaction; lanes 13–20, 1×, 2μg per reaction) using a radiolabeled probe that contains the TdT D′ regulatory element.