Post-Translational Modification of Pdx1 Figure 1. Over expression of Pdx1 in L and HEK293 cells. A) Raw data from the NIA analysis can be visualized as gel-bands or as a graph. B) The intensity of the signal can be estimated based on the height of the peak or the area under the curve. C-E) Over expression of empty vector or pdx1WT in L cells followed by western blotting using a goat-a-Pdx1 (C, top) or a mouse-a-Pdx1 antibody (C, bottom) and NIA analysis of the same lysates using the goat-a-Pdx1 (D) or mouse-a-Pdx1 antibody (E). doi:10.1371/journal.pone.0035233.g001 5.9 peak is the Pdx13xFLAG protein. NIA analysis of Pdx13xFLAG purified from HEK cells resulted in four specific peaks at 5.1, 5.2, 5.3 and 5.5 which are similar to those observed when analyzing the lysates without the IP step (Fig. 2H). As for IP of Pdx1WT, lower intensity bands is also evident for Pdx13xFLAG and we believe that the peaks at pI 5.8 and 5.9 corresponds to the 6.9 and 7.2 peaks found for Pdx1WT. In contrast the 5.6 peak is found in both control and Pdx1 expressing cells and this peak does not move in the response to the 3xFLAG tag, indicating that it is unspecific binding of the goat-a-Pdx1 antibody. Lastly, to rule out that differences in buffer composition following the IP could influence the migration of Pdx1 in the nano capillaries we carried out a series of experiments where the purified Pdx1 from bacteria or mammalian cells where spiked directly into lysates from HEK cells transfected with EGFP or Pdx13xFLAG (Fig. S1). These results strongly indicate that the bacterial Pdx1 generates one single peak with a pI that is different from the Pdx1 expressed in mammalian cells, thus confirming the presence of post-translational modifications on Pdx1 when expressed in mammalian cells. Endogenous Pdx1 also Exist in Multiple Isoforms Having confirmed that we are able to detect multiple isoforms of Pdx1 following over-expression in mammalian cell lines we went on to test if the NIA analysis could be used to detect endogenous Pdx1 protein. We therefore analyzed the Pdx1 profile from various cell types and tissues known to be positive or negative for Pdx1 expression. Another question we wanted to address was to what extent the Pdx1 profile is influenced by protein-protein interactions. Normally, the NIA analysis is conducted in a mild Hepes buffer (HNG) and it is therefore possible that some of the Pdx1 peaks we observe represent a protein complex. We therefore compared the profiles of endogenous Pdx1 in the HNG buffer (Fig. 3A-D) with that obtained in 8 M urea which will denature most interactions (Fig. 3A’-D’). According to the manufactures instruction urea treatment gives better resolution but reduces signal intensity. Using the goat-a-Pdx1 antibody on bTC cells the characteristic 6.0, 6.1 and 6.4 peaks were clearly seen in both buffers (Fig. 3A, 3A’). To a lesser extend also the 5.9 peak and perhaps the 6.3 as a faint shoulder on the left side foot of the 6.4 peak. The 6.9 peak was small but significantly present in HNG buffer only, whereas the 6.5 peak was only observed in the urea treated samples. The peak at 5.8 which was only detected with the goat-a-Pdx1 antibody was present in both buffers while the non-pdx1 peak at 5.6 was only observed with urea. In aTC cells which express little or no Pdx1, the strongest peaks were at pI 5.5 and 5.6 and perhaps also at 6.5 in urea treated lysate (Fig. 3B, 3B’). In embryonic tissue Pdx1 is expressed in the pancreatic endoderm and in three independent lysates from isolated embryonic pancreata we observed a profile very similar to bTC with the strongest peaks at pI 6.0, 6.1 and 6.4 (Fig. 3C, 3C’). This observation was very reproducible in both buffers although again the peaks around 5.6 became more prominent in 8M urea. Lastly, we compared the profile obtained from three independent samples of isolated mouse islets (Fig. 3D, 3D’). Here we observed increased variation between the different samples, but interestingly in the urea treatment revealed a strong peak at pI 6.9 in addition to the characteristic 6.0 and 6.4 peaks. We also detected some smaller peaks around 5.9, 6.1 and 6.8. Lastly, we analyzed the different PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35233