Fig 2: EGFR activation regulates Menin subcellular localization.(A, B) Representative IF images of FLAG-Menin–expressing MEFΔMen1 and AGS cells showing FLAG-WTMenin localization (white) under the indicated treatment conditions. Arrows indicate representative cells exhibiting cytoplasmic redistribution of Menin. Scale bar, 20 μm. (D, E) Quantification of FLAG-Menin subcellular localization in MEFΔMen1 and AGS cells. FLAGC cells were scored per high-power field (HPF) and classified as nuclear (N) or cytoplasmic (C). Data are expressed as the percentage of nuclear (white) and cytoplasmic (gray/black) FLAGC cells per HPF from ≥5 randomly selected fields per condition across ≥3 independent experiments. Images were analyzed at 400× Mag across the experiments. Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple-comparison test. ***P < 0.001. (E, F) Western blot analysis of nuclear and cytoplasmic protein extracts from AGS cells transfected with wild-typw Menin, C490, or C470 constructs with or without EREG and MG132. Lamin B1 (LMNB1) and β-tubulin served as nuclear and cytoplasmic loading controls, respectively. (G, H) Western blot analysis of nuclear and cytoplasmic protein extracts from KATO III cells transfected with wild-type Menin, C490, or C470 constructs following EREG and MG132 treatments. LMNB1 and β-tubulin served as nuclear and cytoplasmic loading controls, respectively. (I) Quantification of FLAG-Menin protein expression in AGS cells normalized to loading controls. (J) Quantification of FLAG-Menin protein expression in KATO III cells normalized to loading controls. Data are presented as mean ± SEM; Two-way ANOVA with Sidak’s post hoc test. ***P < 0.01; (n = 3 biological replicates).