FIGURE 4.

Differential expression of menin mutants is conserved in gastrin-expressing tumor cell lines. A, Western blot analysis of endogenous menin protein expression in MEF^ΔMen1, MEF wild-type, AGS, MKN-45G, KATO III, BON-1, and GLUTag cell lines. B, Western blot analysis of menin and FLAG expression in whole-cell extracts from AGS, MKN-45G, and GLUTag cells following overexpression of empty vector (pcDNA, lane 2), wild-type menin (lane 3), and the three mutated menin proteins (lanes 4–6). Untransfected cells (UT) are shown in lane 1. C, Western blot analysis of FLAG expression in nuclear and cytoplasmic protein extracts in AGS, MKN-45G, and GLUTag cells. 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. D, Quantitation of FLAG protein band intensity in nuclear and cytoplasmic compartments normalized to respective loading controls. n = 3 experimental replicates; ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA with Tukey post hoc test; mean ± SEM. E, Immunofluorescent images of FLAG-stained AGS and MKN-45G with FLAG-Menin shown in white. Scale bar = 10 μm. Quantitation of FLAG expression in nuclear and cytoplasmic compartments expressed as a percentage of positively transfected AGS (F), MKN-45G (G), and GLUTag (H) 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. Relative menin mRNA expression in AGS (I), MKN-45G (J), and GLUTag (K) cells 48 hours following overexpression of menin plasmid constructs. WT, wild-type.