esearch, the cross absorbed versions. For western and NIA they were HRP conjugated and used at 1:10.000 (western) or 1:500 (NIA). For immunofluorescence they were Cy2, Cy3 or Cy5 conjugated and used at 1:300. Supporting Information Figure S1 into HEK293 cells expressing either EGFPFlag or Pdx13xFlag. To further validate that the pI of bacterially expressed Spiking of Pdx13xFLAG purified from bacteria Pdx1 does differ from that of Pdx1 expressed in the human HEK293 cell line, we mixed the purified Pdx13xFLAG protein sample from BL21 bacteria into HEK293 lysate expressing either EGFPFLAG or Pdx13xFLAG and then ran the NIA assay. As control we spiked HEK293 lysates expressing (A) EGFPFLAG or (B) HEK293 lysates expressing Pdx13xFLAG with a negative control sample consisting of bacterial proteins purified from untransformed bacteria. As expected from Fig.3 we only observed the Pdx13xFLAG profile. (C and D) Then we spiked the same two HEK293 lysates with purified bacterially expressed Pdx13xFLAG and it is clear that the profile was not altered by mixing the samples before doing the NIA analysis. Two analyses were performed giving similar results. (TIF) Figure S2 NIA profile of endogenous Pdx1 using the mouse-a-antibody. Samples were diluted in 8 M urea and analyzed by NIA with the mouse-a-Pdx1 antibody. This was done in parallel to the profiles obtained with the goat-a-Pdx1 antibody, presented in figure 3. A-D) NIA analysis showing the profile of bTC (A), aTC (B), E15.5 pancreas (C) and islets (D) obtained with the mouse-a-Pdx1 antibody (red) superimposed on the profile obtained from the same samples using the goat-a-Pdx1 antibody (grey). Two analyses were performed giving similar results. (TIF) References 1. Jonsson J, Carlsson L, Edlund T, Edlund H (1994) Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371: 606–609. 2. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, et al. (1996) PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122: 983–995. 3. Ohlsson H, Karlsson K, Edlund T (1993) IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J 12: 4251–4259. 4. Wright CV, Schnegelsberg P, De Robertis EM (1989) XlHbox 8: a novel Xenopus homeo protein restricted to a narrow band of endoderm. Development 105: 787–794. 5. Jorgensen MC, Ahnfelt-Ronne J, Hald J, Madsen OD, Serup P, et al. (2007) An illustrated review of early pancreas development in the mouse. Endocr Rev 28: 685–705. 6. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF (1997) Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 15: 106–110. 7. Grapin-Botton A, Majithia AR, Melton DA (2001) Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev 15: 444–454. 8. Pedersen JK, Nelson SB, Jorgensen MC, Henseleit KD, Fujitani Y, et al. (2005) Endodermal expression of Nkx6 genes depends differentially on Pdx1. Dev Biol 288: 487–501. 9. Horb ME, Shen CN, Tosh D, Slack JM (2003) Experimental conversion of liver to pancreas. Curr Biol 13: 105–115. 10. Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H (1998) beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev 12: 1763–1768. 11. Johnson JD, Ahmed NT, Luciani DS, Han Z, Tran H, et al. (2003) Increased islet apoptosis in Pdx1+/- mice. J Clin Invest 111: 1147–1160. 12. Hani EH, Stoffers DA, Chevre JC, Durand E, Stanojevic V, et al. (1999) Defective mutations in the insulin promoter factor-1 (IPF-1) gene in late-onset type 2 diabetes mellitus. J Clin Invest 104: R41–48. 13. Stoffers DA, Ferrer J, Clarke WL, Habener JF (1997) Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet 17: 138–139. Post-Translational Modification of Pdx1 Figure S3 The Pdx1 protein is detected in developing mouse endoderm. Immunohistochemical stainings showing the Pdx1 expression (green) in the Chd1 positive endoderm (red). At e10.5 (A) and e12.5 (B) Pdx1 is expressed uniformly in the pancreas, posterior stomach and in the duodenum. A’ and B’) Pdx1 NIA analysis of equivalent micro dissected tissue, showing that during very early development at e10.5 Pdx1 also appears to show the characteristic NIA profile and two days later at e12.5 the profile is easily recognizable. Results are representative of three independent experiments. (TIF) Figure S4 Both Pdx1WT and Pdx1S61A induces ectopic insulin expression in vivo. 3D image projections of confocal sections obtained from chicken embryos stained for Insulin (red), Nkx6.1 (blue) and GFP (green) by whole mount immunohisto- chemistry 72 hours after electroporation. In embryos electropo- rated with plasmids encoding (A) Pdx1WT or (B) Pdx1S61A we observe an equal amount of ectopic insulin production. Two analyses were performed giving similar results. (TIF) Acknowledgments We thank Dr. Chris Wrigh for providing the goat-a-Pdx1 antibody and Gsk3b plasmid, Dr. Anne Grapin-Botton for providing the pCGIG5 plasmid expression vector, and Dr. Gerard Gradwohl for the Neurog3 mice. We are grateful to Dr. Palle Serup and Dr. Ole D. Madsen for discussions and critical comments, Søren Refsgaard Lindskog for FACS analysis and Hanne Duus Laustsen for technical assistance. Author Contributions Conceived and designed the experiments: TF KBS SK MLN JH-S. Performed the experiments: TF KBS JH-S. Analyzed the data: TF KBS SK MLN JH-S. Contributed reagents/materials/analysis tools: TF KBS MLN JH-S. Wrote the paper: TF JH-S. 14. Huang H, Liu N, Lin S (2001) Pdx-1 knockdown reduces insulin promoter activity in zebrafish. Genesis 30: 134–136. 15. Leibowitz G, Ferber S, Apelqvist A, Edlund H, Gross DJ, et al. (2001) IPF1/ PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal With type 2 diabetes. Diabetes 50: 1799–1806. 16. Milewski WM, Duguay SJ, Chan SJ, Steiner DF (1998) Conservation of PDX-1 structure, function, and expression in zebrafish. Endocrinology 139: 1440–1449. 17. Seufert J, Weir GC, Habener JF (1998) Differential expression of the insulin gene transcriptional repressor CCAAT/enhancer-binding protein beta and transactivator islet duodenum homeobox-1 in rat pancreatic beta cells during the development of diabetes mellitus. J Clin Invest 101: 2528–2539. 18. Yee NS, Yusuff S, Pack M (2001) Zebrafish pdx1 morphant displays defects in pancreas development and digestive organ chirality, and potentially identifies a multipotent pancreas progenitor cell. Genesis 30: 137–140. 19. Holland AM, Gonez LJ, Naselli G, Macdonald RJ, Harrison LC (2005) Conditional expression demonstrates the role of the homeodomain transcription factor Pdx1 in maintenance and regeneration of beta-cells in the adult pancreas. Diabetes 54: 2586–2595. 20. Waeber G, Thompson N, Nicod P, Bonny C (1996) Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol Endocrinol 10: 1327–1334. 21. Watada H, Kajimoto Y, Umayahara Y, Matsuoka T, Kaneto H, et al. (1996) The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells. Diabetes 45: 1478–1488. 22. Chakrabarti SK, James JC, Mirmira RG (2002) Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding. J Biol Chem 277: 13286–13293. 23. Kulkarni RN, Jhala US, Winnay JN, Krajewski S, Montminy M, et al. (2004) PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance. J Clin Invest 114: 828–836. 24. Fujimoto K, Chen Y, Polonsky KS, Dorn GW 2nd (2010) Targeting cyclophilin D and the mitochondrial permeability transition enhances {beta}-cell survival and prevents diabetes in Pdx1 deficiency. Proc Natl Acad Sci U S A. PLoS ONE | www.plosone.org 14 April 2012 | Volume 7 | Issue 4 | e35233