Figure 7. The Pdx1 NIA profile from in vivo β-cells is not unique and not responsive to glucose.

To determine if the β-cells contain a unique Pdx1 protein species we compared the NIA profiles of pancreata obtained from wild type mice and mice lacking the pro-endocrine gene Neurog3. In the Neurog3^−/− mice all β-cells are lost and the Pdx1 high expressing β-cells are no longer present. Thus, any modifications on Pdx1 that are uniquely present in the β-cell should therefore only appear in the wild type profile. A and B) Immunohistochemical stainings of e15.5 Neurog3^+/+ or Neurog3^−/− mouse pancreata, showing the distribution of Pdx1 (green) in the Chd1 positive endoderm (red). A’ and B’) the Pdx1 NIA analysis of equivalent micro dissected tissue. Pdx1 NIA profile of pancreas tissue lysate from newborn (P2) (C) and adult NMRI mice (D). Results are representative of two (Neurog3) or three independent (P2 and adult) experiments. E, F and G) Three independent purifications of islets from NMRI mice were cultured for 2–3 days, washed with media lacking glucose, and then cultured with 2 mM glucose for two hours followed by incubation with 2 mM or 30 mM glucose for one hour E) Western blot on media from the islets, showing glucose responsiveness. F) Three preparations of mouse islets in 2 mM glucose. G) Three preparations of mouse islets in 30 mM glucose. H) Quantification of the three experiments showing the ratio of the pI 6.0 peak area under curve divided by the pI 6.1 peak area under curve. The error bars show standard deviation.