Post-Translational Modification of Pdx1 Figure 2. The Pdx1 NIA profile differs between bacterial and mammalian cells. First, a comparison of the basal NIA profile of pdx1WT and pdx13xFLAG protein from transfected HEK293 cells by western blotting (A) and NIA (B), employing the goat-a-Pdx1 antibody. The amino acids in the 3xFLAG tag are negatively charged and in agreement a left shift of the NIA profile is observed. Results are representative of at least three independent experiments. Then a comparison of purified Pdx1WT and Pdx13xFLAG protein from E.Coli BL21 cells versus purified protein from HEK293 cells. Plasmids coding for the indicated proteins were expressed in BL21or HEK293 cells and purified by immunoprecipitation using a monoclonal mouse-a-Pdx1 antibody coupled to protein G beads (Pdx1WT) or with anti-FLAG beads (Pdx13xFLAG). As negative control cell lysate from non transformed bacteria or empty vector (EV) transfected HEK293 cells were ran in parallel. Purified protein from Pdx1WT expressing and non expressing BL21 and HEK293 cells were analyzed by coomassie staining and western blot (C) and by NIA, (D) BL21, (E) HEK293. F-H) Similar analysis for Pdx13xFLAG. These experiments were performed at least twice obtaining similar results. doi:10.1371/journal.pone.0035233.g002 samples with the mouse-a-Pdx1 antibody (Fig. S2), confirming the peaks at 6.0, 6.1 and 6.4 as well as the islet enriched 6.9 peak. In summary the NIA analysis of three cell types known to express endogenous Pdx1 produced several peaks which varied slightly between the different cell types. However, the overall profile is very similar to that obtained using over-expression. Both Endogenous and Exogenous Pdx1 Carry One Principal Phosphorylation Depending on expression levels and cell types we detect at least four or five differentially charged isoforms of Pdx1 in various cell types and tissues while bacterially generated Pdx1 only produces a single peak. This strongly suggests that Pdx1 is subject to post translational modifications in mammalian cells. Numerous studies have shown that Pdx1 is phosphorylated [25,26,27,28,29,30,31,32,33,34] and to PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35233