FIGURE 2.

Tumor-acquired mutations in MEN1 result in aberrant subcellular localization of menin at the protein level. Empty vector (pcDNA), wild-type menin, and menin mutant proteins bearing FLAG-epitope tags were transiently overexpressed in Men1-null mouse embryonic fibroblasts (MEF^ΔMen1). A, Western blot analysis of nuclear and cytoplasmic protein extracts probed for FLAG expression. Nuclear fractions are shown in lanes 1, 3, 5, 7, and 9 while cytoplasmic fractions are shown in lanes 2, 4, 6, 8, and 10. Histone H3 and β-tubulin were used as loading control markers for nuclear and cytoplasmic fractions, respectively. B, Quantitation of FLAG protein band expression in nuclear and cytoplasmic compartments normalized to respective loading controls. n = 3 experimental replicates; **, P < 0.01; ***, P < 0.001 by two-way ANOVA with Tukey post hoc test; mean ± SEM. C, Immunofluorescent images of FLAG-stained MEF^ΔMen1cells (red) costained with DAPI to visualize the nucleus (blue). Bottom panel shows FLAG staining in white. Scale bar = 10 μm. D, Quantitation of FLAG expression in nuclear and cytoplasmic compartments expressed as a percentage of positively transfected cells. Ten images taken at 200X magnification were counted across n = 3 experiments for a total of 30 images per group; *, P < 0.05 by two-way ANOVA with Tukey post hoc test; mean ± SEM. E, Relative Men1 mRNA expression in menin-null MEFs following overexpression of empty vector, wild-type menin, and the three mutants. F, Relative Men1 mRNA expression in wild-type MEFs expressing endogenous menin as a control. n = 3–4 experimental replicates; n.s. by one-way ANOVA. WT, wild-type.