IKAROS transcripts and protein expression in patients. (A) The chromatograms show the sequence of the indicated heterozygous mutation in complementary DNA prepared from peripheral blood mononuclear cells (PBMCs). WT indicates WT of IKAROS’ reference sequence. Arrows indicate the sites of mutations. (B) IKAROS protein expression levels were tested in T and B cells from PBMCs from index patients and a paired HD control. (C) IKAROS expression in nuclear extracts of T-cell blasts. Triangles indicate R213* and S427* mutant proteins. The lower blots were overexposed to visualize the low levels of truncated IKAROS protein (R213* and S427*). Data are representative of 2 independent experiments. (D-E) HEK293T cells were transfected with a vector expressing IKAROS WT or mutant. After 24 hours, cells were treated with cycloheximide (CHX; 20 μg/mL) for an additional 24 hours. (D) Whole-cell lysates were prepared and analyzed by immunoblot analysis using hemagglutinin (HA) antibodies to test IKAROS expression. Vinculin was used as a loading control. (E) The relative IKAROS protein stability was calculated by dividing the CHX-treated sample by the untreated sample (×100) after normalization with vinculin to show the remaining protein amount after CHX treatment. Data are mean ± standard error of the mean of 3 or 4 independent experiments. Data (WT vs each mutant) were analyzed using GraphPad Prism software. *P < .05, unpaired Student t test.