Dominant-negative effects of variant ETV6 on WT ETV6. (A) ETV6 transcription repressor activity was measured by cotransfecting HEK293T with constructs encoding WT ETV6, increasing amounts of variant ETV6, and a luciferase reporter. The top panel denotes missense variants, while the bottom panel indicates nonsense or frameshift variants. Bars show the mean of triplicate experiments and repeated at least 3 times; error bars represent SD. **P < .01 in ≥1 condition. (B) HEK293T cells were cotransfected with MYC-tagged WT and FLAG-tagged variant ETV6 constructs followed by pull-down with anti-FLAG beads for western blotting to examine dimerization. Seven representative variants were chosen for this assay. IP, immunoprecipitation. (C) HEK293T cells were cotransfected with MYC-tagged WT and the same 7 FLAG-tagged variant ETV6 constructs as in panel B followed by subcellular fractionation and western blotting of nuclear and cytoplasmic fractions using an anti-MYC antibody. Red bars indicate frameshift variants, orange bar indicates a nonsense variant, and lime green bars indicate missense variants. Bars represent the nuclear-to-cytoplasmic ratio from triplicate experiments; error bars represent SD. *P < .05. In panels A and C, a 1-way ANOVA with multiple comparison was performed to compare each variant with WT.