DNA binding and transcription activity of the mutant IKAROS protein. (A-B) EMSAs were performed using nuclear extracts from HEK293T cells transfected with the indicated IKAROS mutation alone (A) or together with WT IKAROS (B). Numbers indicate the ratio of WT and mutant IKAROS DNA used for the cotransfection. The nuclear extracts were allowed to bind to 2 IKAROS probes: IK-bs1, an IKAROS consensus binding sequence, and γ-Sat 8, a sequence from the pericentromeric region of human chromosome 8. IKAROS-containing complexes are indicated with arrows. Triangles indicate mutant IKAROS protein binding to the DNA as a monomer. Data are representative of 3 independent experiments. (C) Four repeats of IKBS1 were inserted into the pGL4.11 vector (pGL4.11-IKBS1) and cotransfected with pcDNA3-HA IKAROS WT or mutants and pRL-TK (Renilla luciferase) as indicated. Twenty hours later, cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System. Firefly luciferase activity was normalized to Renilla luciferase activity, and the results were normalized to the empty vector (EV) control. Each experiment was carried out in duplicate. Data are mean ± standard error of the mean from 3 independent experiments.