in chicken embryos electroporated with Pdx1S61A and Pdx1S61E (Fig. 8G, 8H). We found that both mutants induces pancreas budding and Nkx6.1 expression to exactly the same extent as the wildtype and it is therefore unlikely that phosphorylation of S61 has a role in pancreas development. In embryos electroporated with Pdx1WT the ectopic Nkx6.1 was found scattered within the Foxa2 positive endoderm. However, in several cases we observed that the Nkx6.1 cells became organized into a branched epithelium very similar to the endogenous pancreas (arrows in Fig. 8F-8H). We therefore speculated that the ectopic Nkx6.1 expression might actually reflect the formation of ectopic pancreata. To test this we stained embryos electroporated with Pdx1WT or Pdx1S61A for GFP, Nkx6.1 and insulin. In all embryos where we could detect ectopic Nkx6.1 expression anterior to the pancreas we also observed insulin expression (Fig. S4A and S4B). Discussion One advantage of the NIA technology is that one antibody is used for the detection of all protein species which can be separated by electrical charge. In recent years NIA has been used successfully to study signal transduction [41,42] but, to the best of our knowledge this work represents the first study where the technology has been applied to a transcription factor. When analyzing the profile of the homeobox containing transcription factor Pdx1 we find at least four differently charged protein species Pdx16.0, Pdx16.1, Pdx16.3 and Pdx16.4. These data were obtained using two different antibodies against Pdx1 and we were able to shift the peaks by adding an epitope tag. Furthermore, the four peaks are always present when Pdx1 is expressed in eukaryotic cells (bTC, mouse islets, mouse embryonic pancreas, L cells, HEK293, aTC and chicken endoderm cells) while bacterially derived Pdx1 only produces a single peak. Given the similar SDS- PAGE mobility of eukaryotic and bacterial Pdx1 we conclude that the NIA peaks we observe are due to post-translational modifications. Also, as both eukaryotic and bacterially expressed Pdx1 gives a single band using western blots we conclude that the post-translational modifications must be relatively small, i.e. under 1000 Da. One very important aim of this study was to determine the pI of bacterially expressed and hereby minimally modified Pdx1 protein and to this end confirm that the Pdx1 protein is in fact subjected to post-translational modification in eukaryotic cells. We did this by comparing the NIA profiles of Pdx1 purified from bacteria and mammalian cells. As expected the bacterial protein produced a single peak. However, the bacterial peak has a pI of 7.2 while the predicted pI for unmodified mouse Pdx1 is 6.4. In lysates from eukaryotic cells the most prominent peak actually runs at 6.4, importantly when comparing bacterial and mammalian Pdx1 we used exactly the same open reading frame for expression and we sequence verified the different expression vectors and can conclusively rule out any possibility of errors in the constructs or the presence of alternative start sites. We therefore conclude that the pI for unmodified Pdx1 is pI 7.2 and that it is a coincidence that the 6.4 peak observed in eukaryotic cells has the same pI as predicted for unmodified Pdx1. This is supported by NIA analysis of human pdx1, which has a predicted pI of 7.1, but when over expressed in HEK293 cells gives the dominant peak at pI 6.6 (data not shown). Interestingly, on western blots Pdx1 also behaves differently from what would be expected. The predicted molecular weight of Pdx1 is 31 kDa but on gels the protein migrates at 40– 46 kDa. This difference has now been proven by mass spectrometry to be due to anomalous SDS-PAGE mobility of the pdx1 protein [45] and we speculate that similar anomalous Post-Translational Modification of Pdx1 properties of Pdx1 in isoelectric focusing is the reason for the difference between observed and predicted pI of bacterially expressed Pdx1 protein. Using the NIA assay and mass spectrometry we identified S61 as the primary phosphorylation and that the modification is responsible for the 6.0 peak. However, the mass spectrometry also identified phosphorylations on Y140/T141, S269 and in the NIA profile we could still detect a smaller peak at 6.0 in the S61A mutant that could be shifted following de-phosphorylation. To test if Y140/T141 orS269 could account for the residual phosphor- ylation in Pdx1S61A we generated the following double mutants Pdx1S61A/Y140A, Pdx1S61A/T141A Pdx1S61A/S269A. However, all the double mutants showed low levels of phosphorylation (data not shown). Given the numerous publications describing other phosphorylation sites on Pdx1 it is obvious that any of those could be present together with the S61 phosphorylation. Given the dynamic and often fast exchange of phosphorylation and post-translational modifications in general it is remarkable that there is so little variation between Pdx1 obtained from the very different cell types. Both endogenous and ectopically derived Pdx1 produces profiles where the 6.0 and 6.4 peaks represent the predominant protein species. Subtle variations might be found, for example the 6.1 peak appears to be more abundant in the E15.5 embryonic tissue. However, only in isolated islets do we observe a significantly different peak at pI 6.9. This peak is interesting for several reasons. First, it is only observed following treatment with 8 M urea indicating that in HNG buffer this Pdx1 species is masked from antibody detection. A likely explanation is that that the 6.9 species only exists in a tight protein-protein complex. Second, the 6.9 peak is only found in isolated islets strongly suggesting that it is unique to mature beta-cells. It should be noted that that the 6.9 peak, was absent in post-natal and adult pancreas, but this is probably due to the expression of Pdx1 in acinar cells, masking the Pdx1 signal from the relatively few b-cells present in the adult mouse pancreas [46]. What our data clearly shows is that the level of glucose has no impact the NIA profile of Pdx1 from purified mouse islets. This is in agreement with other studies also using non-invasive detection techniques to study Pdx1 protein from both mouse, human and rat islets [29,45]. One explanation for this could be that the factors modifying Pdx1 are ubiquitous. For example if the posttransla- tional modifications of Pdx1 were coupled to common events such as the cell cycle [47]. Alternatively, ubiquitous factors known to interact with Pdx1 such as p300 [48] might be present in all cell types and therefore influence the profile in a similar manner. One important feature of the NIA assay is that can be used to study how the different modifications influence each other. Although we fail to identify the modification responsible for the 6.3 and 6.4 peaks it is very obvious that these peaks are uncoupled from the phosphorylation of S61. Thus the de-phosphorylation only affects the 6.0 and 6.1 peaks. Furthermore, S61E which mimics a constitutive phosphorylation abolished other protein isoforms. One explanation for this could be that the modification responsible for the 6.3/6.4 peaks only can occur when S61 is not phosphorylated. Likewise the residual phosphorylation peak (6.0) in Pdx1S61A is remarkably larger than what would be expected if the secondary phosphorylation peak (5.9) of the wild type Pdx1 protein was shifted to (6.0). Thus interactions between different modifications or redundant phosphorylation sites are likely to influence the dynamics of Pdx1 modification. In conclusion, we show that Pdx1 harbors several post- translational modifications and that phosphorylation of serine 61 PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e35233