Figure 4. Gastrin induced nuclear export leads to its proteasomal degradation in the cytoplasm.

(A) IF staining of menin in glial cultures isolated from duodenal lamina propria of C57 WT mice and treated without or with gastrin (20 nM, 8 h) in the presence or absence of Leptomycin b (LMB, 10 μM), or MG132 (25 μM). (B) Representative blots showing menin expression in nuclear and cytoplasmic fractions of glial cultures from C57 WT mice and treated as in (a). (C) IF staining of menin and ubiquitin in glial cultures isolated from C57 WT mice treated with or without gastrin (20 nM, 8 h) in the presence or absence of MG132 (25 μM). (D, E) Representative blots showing gastrin-induced menin ubiquitination in STC-1 cells. STC-1 cells were treated without or with gastrin (20 nM) for 0, 2, or 4 hours in the presence or absence of MG132 (25 μM). Menin was immunoprecipitated (IP) from the cytoplasmic fraction. Immunoblot (IB) for poly-Ub (top panel), and IB for menin (lower panel). Menin versus Ub-menin indicated. Blot for IgH and GAPDH in input lysates are loading controls (D). Ubiquitinated proteins were immunoprecipitated from the cytoplasmic fraction and detected with Ub antibodies (E). IB for poly-Ub (top panel), and Ub-menin (lower panel). (F) Representative blot (top) and quantitation (bottom) showing menin expression from Cycloheximide (CHX) chase experiment in STC-1 cells (n= triplicates from 4 separate experiments). STC-1 cells were treated with or without gastrin in the presence or absence of MG132 (25 uM) in the presence of CHX (25μg/mL) for 0, 2, 4, and 8 hours. Data shown are the Mean ± SEM. * p< 0.05, ** p< 0.01. Scale bars: (A, C) – 20 μm.