Post-Translational Modification of Pdx1 Figure 3. The NIA profile of endogenous Pdx1. Analysis of cells with know endogenous expression of Pdx1. As negative control we included aTC cells which express little or no Pdx1. To rule out the possibility of protein-protein complexes affecting the NIA profile, the lysates were diluted 20 fold in a mild Hepes buffer (HNG) (A-D) or in 8 M urea (A’-D’) prior to NIA analysis. Using the goat-a-Pdx1 antibody, the characteristic 6.0, 6.1 and 6.4 peaks and to a lesser extend also the 6.3 peak is observed in bTC (A, A’) but not in aTC (B, B’). To show the reproducibility of the NIA assay between the HNG and 8 M urea we compared the Pdx1 profile from e15.5 mouse pancreas obtained from three different litters (C, C’) and from 3 different preparations of purified mouse islets (D, D’). We also performed these analyses with the mouse-a-Pdx1 antibody (Fig. S2), confirming the dominant peaks at 6.0, 6.1 and 6.4 as well as the islet enriched 6.9 peak. doi:10.1371/journal.pone.0035233.g003 PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35233