Post-Translational Modification of Pdx1 Figure 7. The Pdx1 NIA profile from in vivo b-cells is not unique and not responsive to glucose. To determine if the b-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 Neurog32/2 mice all b-cells are lost and the Pdx1 high expressing b-cells are no longer present. Thus, any modifications on Pdx1 that are uniquely present in the b-cell should therefore only appear in the wild type profile. A and B) Immunohistochemical stainings of e15.5 Neurog3+/+ or Neurog32/2 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. doi:10.1371/journal.pone.0035233.g007 detect the 6.0, 6.1 and 6.4 peaks indicating that Pdx1 is modified during the earliest stages of pancreas development. Slightly later in development at e12.5 the pancreas express high levels of Pdx1 while low Pdx1 expression can be detected in the stomach and duodenum (Fig. S3B). Again we found the distinctive Pdx1 profile albeit at higher levels (Fig. S3B’). To test if the phosphorylated form of Pdx1 is important for pancreas development we electroporated mouse Pdx1 into the endoderm of living chicken embryos using in ovo electroporation [44]. Using this system Pdx1 would be expressed in an environment closely resembling the developing pancreas and presumably be exposed to the similar signaling cues. One day after electroporation GFP expression can be detected throughout the endoderm (Fig. 8A). The embryos were then dissociated into single cells and sorted into GFP negative and GFP positive fractions using FACS. On average GFP cells constituted 0.3% of the total number of cells (pre-sort) and 53% after sorting. Each embryo contained 5000–10000 GFP express- ing cells. Running NIA analysis on cells from embryos electropo- rated only with GFP, the GFP sorted fraction was positive for GFP but not Pdx1 (Fig. 8B), showing that we do not detect endogenous chicken Pdx1. In the NIA analysis of embryos co-electroporated with GFP and Pdx1 neither of the two proteins were detected in the GFP negative fraction (Fig. 8C), but only in the GFP positive fraction (Fig. 8D). As expected, the Pdx1 profile produced peaks at pI 6.0 and pI 6.4 confirming that Pdx1 becomes phosphorylated in the developing endoderm. Finally, we electroporated chicken embryos with plasmids encoding GFP, Pdx1WT, Pdx1S61A or Pdx1S61E. The embryos were allowed to develop for 3 days, before PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35233