Functional characterization of ERG variants. (A) ERG variants residing in the ETS domain reduce transactivation. K562 cells were transfected with pcDNA3 empty vector (EV) or pcDNA3-ERG (WT or variants). All constructs were cotransfected with a luciferase reporter plasmid driven by an ITGA2B promoter (quadruplicate replicates, repeated 3 times). Fold change (mean ± standard error of the mean [SEM]) in comparison with the WT is plotted. Pairwise comparisons are shown (∗P < .05 in comparison with WT). (B) ETS domain variants reduce ERG DNA binding affinity. EMSA of WT ERG and variants. Transfected HEK293 whole cell lysates were prepared and bound to an oligonucleotide containing an ETS DNA consensus sequence. Probes were visualized using chemiluminescence. Pairwise comparisons are shown (∗P < .05 compared with WT). (C) ERG ETS domain variants alter subcellular localization. A Myc-tag was added to WT ERG and variants. COS-7 cells were transfected with pcDNA3 EV, pcDNA3-ERG-Myc (WT), and pcDNA3-ERG-Myc variants. Cells were stained for a Myc-tag and DAPI (4′,6-diamidino-2-phenylindole). The nuclear to cytoplasmic ratio of each variant was quantified and the fold change (mean ± SEM) in comparison with the WT was plotted. Pairwise comparisons are shown (∗P < .05 compared to WT). Nuclear to cytoplasmic ratio was obtained from 3 independent experiments. In all comparisons, a Student t test was used (∗P < .05; ∗∗P < .01; ∗∗∗∗P < .001 in comparison with WT).