Novel monoclonal antibodies against Pdx1 reveal feedback regulation of Pdx1 protein levels

  • T. Galbo Hagedorn Research Institute, Department of Beta-cell Regeneration, Denmark.
  • I. L Pedersen Novo Nordisk A/S, Department of Antibody Technology, Denmark.
  • T. Fløyel Hagedorn Research Institute, Department of Diabetes Genome Biology, Denmark.
  • C. H. Bang-Berthelsen Hagedorn Research Institute, Department of Diabetes Genome Biology, Denmark.
  • P. Serup Hagedorn Research Institute, Department of Beta-cell Regeneration, Denmark.
  • O. D Madsen Hagedorn Research Institute, Department of Beta-cell Regeneration, Denmark.
  • J. Hald | hald@hagedorn.dk Hagedorn Research Institute, Denmark.

Abstract

The aim of this study was to characterize two monoclonal antibodies (F6A11 and F109-D12) generated against Pdx1 (pancreatic and duodenal homeobox-1), a homeodomain transcription factor, which is critical for pancreas formation as well as for normal pancreatic beta cell function. For production of monoclonal antibodies, we immunized Robertsonian POSF (RBF)mice with a GST-Pdx1 fusion protein containing a 68-amino acid C-terminal fragment of rat Pdx1. These monoclonal antibodies detect Pdx1 by western blotting and allow immunohistochemical detection of Pdx1 in both mouse and rat tissue. F6A11 and F109-D12 produce IHC staining patterns indistinguishable from that obtained with highly specific polyclonal Pdx1 antisera raised in rabbits and goats, when applied to embryonic or adult mouse pancreatic tissue. In contrast to previously generated polyclonal anti-Pdx1 antisera, we also demonstrate that F6A11 works for intracellular fluorescence activated cell sorting (FACS) staining of Pdx1. By using F6A11, we characterize the induction of Pdx1 in the Doxycycline (DOX) inducible insulinoma cell line INSrαβ-Pdx1 and follow the reduction of Pdx1 after removing Dox. Finally, we show that induction of exogenous Pdx1 leads to a reduction in endogenous Pdx1 levels, which suggests that a negative feedback loop is involved in maintaining correct levels of Pdx1 in the cell.

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Author Biographies

T. Galbo, Hagedorn Research Institute, Department of Beta-cell Regeneration

Department of Beta-cell Regeneration

MS

I. L Pedersen, Novo Nordisk A/S, Department of Antibody Technology

Department of Antibody Technology

PhD

T. Fløyel, Hagedorn Research Institute, Department of Diabetes Genome Biology

Department of Diabetes Genome Biology

MS


C. H. Bang-Berthelsen, Hagedorn Research Institute, Department of Diabetes Genome Biology

Department of Diabetes Genome Biology

MS

P. Serup, Hagedorn Research Institute, Department of Beta-cell Regeneration

Department of Beta-cell Regeneration

PhD

O. D Madsen, Hagedorn Research Institute, Department of Beta-cell Regeneration

Department of Beta-cell Regeneration

PhD

J. Hald, Hagedorn Research Institute

Department of Beta-cell Regeneration

PhD

Published
2010-04-15
Section
Original Papers
Supporting Agencies
NIDDK, Danish Diabetes Association, EU 6th Framework Programme
Keywords:
Pdx1, Pancreas, Diabetes, Antibody, FACS, INSralphabeta-Pdx1, Dox
Statistics
Abstract views: 534

PDF: 277
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How to Cite
Galbo, T., Pedersen, I. L., FløyelT., Bang-Berthelsen, C. H., Serup, P., Madsen, O. D., & Hald, J. (2010). Novel monoclonal antibodies against Pdx1 reveal feedback regulation of Pdx1 protein levels. European Journal of Histochemistry, 54(2), e19. https://doi.org/10.4081/ejh.2010.e19