Post-Translational Modification of Pdx1 Figure 6. MS analysis identifies serine 61 as the principal phosphorylation site in Pdx1. To substantiate the NIA data we did mass spectometry to identify modified residues especially looking for phosphorylations. We analyzed endogenous Pdx1 protein from Min6 cells and over expressed Pdx1 from HEK293 cells. The mouse wildtype Pdx1 protein was, purified by immunoprecipitation using the mouse-a-Pdx1 antibody, then separated by SDS-PAGE, digested with chymotrypsin and subjected to MS analysis. A-B) MS/MS data showing phosphorylation on wildtype Pdx1 at serine 61 when over experessed and purified from HEK293 cells (A) or from Min6 cells (B). We then did a quantification of the amount of phosphorylated versus non-phosphorylated Pdx1 peptides counted by the MS instrument. C) Relative amount of phosphorylated serine 61 and serine 269 in HEK293 and Min6 cells. The MS analysis was done once. doi:10.1371/journal.pone.0035233.g006 cells because they express higher levels of Pdx1 (Fig. 7A; white arrows). In mice lacking the pro-endocrine gene Neurog3 the endocrine lineage is absent and as a result the high expressing Pdx1 cells are no longer be detected (Fig. 7B). To test if the Pdx1 profile from the high expressing b-ell population has any unique features we dissected and homogenized whole pancreata from wild type and Neurog3 deficient e15.5 embryos and subjected the lysates to NIA analysis. As for all other cell types analyzed we detected the 6.0, 6.1 and 6.4 peaks and we could not detect any obvious differences between the wild type and the pancreata lacking b-cells (Fig. 7A’, 7B’). Next we examined the NIA profile from pancreas lysates of newborn (Fig. 7C) and adult (Fig. 7D) mice pancreata to see if any changes could be associated with the b-cells becoming functional and being exposed to fluctuating blood glucose levels. At both P2 and in adult mice the 6.0, 6.1 and 6.4 peaks were indistinguishable from the previously observed Pdx1 profiles including embryonic tissue suggesting that the Pdx1 profile is relatively stable after birth. To test if changes in glucose levels had any effect on the NIA profile we isolated mouse islets and incubated them at low or high glucose (2 mM and 30 mM) for 1 hour, before harvest. We confirmed that the islets were glucose responsive by measuring the amount of insulin released to the media using western blotting (Fig. 7E). However, the NIA analysis shows the profiles from three independent experiments in 2 mM or 30 mM glucose, respectively (Fig. 7F, 7G). It is evident that the profiles obtained from high and low glucose is remarkably similar and when the ratio between the 6.0 peak and the 6.1 peak is calculated there is no difference between the two conditions (Fig. 7H). Mouse Pdx1 is Modified in the Developing Endoderm of Mouse and Chicken Embryos In addition to its role in the b-cell Pdx1 is also required for pancreas development during embryogenesis. At e10.5 Pdx1 is expressed in both the dorsal and ventral pancreatic buds, in the duodenum and in the posterior stomach (Fig. S3A). The stomach and pancreas region from wild type embryos was micro dissected and the pooled pancreata from two litters were analyzed by NIA (Fig. S3A’). The signal obtained is weak but we could robustly PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35233