GST-Pdx1 is of expected size and the monoclonal antibodies F6 and F109 recognize Pdx1 from βTC6 with correct size; furthermore, F6 and F109 specifically recognize Pdx1 in adult mouse pancreas. (A) SDS PAGE analysis of the purified GST-Pdx1 fusion protein, demonstrating the size and purity of the antigen. For the non-reduced protein a double band around 37 kDa is observed (lane 1), whereas under reducing conditions only a single band around 38 kDa is seen (lane 2). In addition, a minor band of approximately 70 kDa is present, which may represent a dimer (filled-in arrowhead). Western blotting of the GST-Pdx1 using the monoclonal anti-Pdx1 antibody F6 revealed that both bands seen on the SDS PAGE gel were derived from the Pdx1 fusion protein (lane 3). The hollow arrow head point to the 70 kDa that is also recognized by F6. (B) Western blot analysis of the purified Pdx1 antibodies F109 (lane 1 and 2) and F6 (lane 3 and 4) for binding to nuclear extracts from βTC6 cells (lane 2 and 4) and αTC1.9 cells (lane 1 and 3) at 10 ug/well, demonstrating that the anti-Pdx1 antibodies identify a single band around 39 kDa corresponding to the Pdx1 protein. This binding could be absorbed with the GST-Pdx1 protein, but not with an irrelevant GST fusion protein (data not shown). (C, D) Immunohistochemical staining of frozen adult mouse pancreas with the purified monoclonal antibodies F6 (C, E and G) and F109 (D, F and H) result in nuclear reactivity in the endocrine islets of Langerhans (C and D). This staining was absorbed by preincubation of the antibodies with the GST-Pdx1 fusion protein – islet is outlined by broken line (E and F), but not by an irrelevant GST fusion protein (G and H). The magnification of histochemical stainings is the same. Scale bar is 50 µm.

Dynamic induction and negative feedback regulation of Pdx1 levels in INSrαβ-Pdx1. The induction of Pdx1 in INSrαβ-Pdx1 cells is followed by FACS analysis using the monoclonal antibody F6 (A, B, and C). A rapid induction of Pdx1 by Dox can be seen with a clear increase in fluorescence intensity from 0 hours (red line) to 4 hours (blue line) before the Pdx1 level peaks after 24 hours (purple line) (A). Following removal of Dox, the Pdx1 level slowly begins to decline, which can be seen after 28 hours (red line) (4 hours after removal of Dox) (B). The Pdx1 level continues to decline from 28 hours (red line) to 32 hours (blue line) (8 hours after removal of Dox), before eventually reaching the lowest point after 48 hours (purple line) (24 hours after removal of Dox) (B). Slowly, from 48 hours (purple line (B)) to 60 hours (red line (C)) (36 hours after removal of Dox) and through to 72 hours (blue line (C)) (48 hours after removal of Dox), the Pdx1 level recovers and gradually approaches the initial level found at 0 hours (red line (a)). Panel a–c constitutes a single experiment, representative of three independent experiments. The bars in figure G represent averages of the relative Pdx1 levels found in the three independent experiments. Student's T-test assuming unequal variances was applied to test whether Pdx1 levels at the time points were different to the level at 0 hours“*” or 24 hours (time of Dox removal)“#”. *P<0.05, **P<0.01, #P<0.05, ##P<0.01, ###P<0.005. Confocal microscopical analysis of the samples analysed on the FACS and later supplied with DNA binding fluorophore DAPI confirm the FACS data (D, E and F). The images reveal a visible increase in Pdx1 from 0 hours (D) to the peak at 24 hours (E) and clear decline to 48 hours (F). Error bars are sd.

Monoclonal immunoreactivity co-localize with immunoreactivity generated by validated anti-Pdx1 sera on sections and in whole mount IHC, and co-localize with β-cells, but not α-cells. Staining of adult pancreas with the monoclonal anti-Pdx1 antibodies F6 (B) and F109 (E) showed a high degree of overlap with the previously described 1858.5 anti-Pdx1 antiserum (A and D), as seen in C and F (double exposures). Whole-mount triple staining of the e10.5 budding stage of pancreas development (G–I) by confocal microscopy: Polyclonal goat anti-Pdx1 (green channel, G); F6 monoclonal anti-Pdx1 (red channel, H); guinea-pig anti glucagon (blue channel in I). Again almost complete immunoreactive overlap in the pancreatic domain is observed between the monoclonal F6 and the goat anti-Pdx1 antiserum (H vs G) – also visualized as yellow in overlay (I). It is also evident that all glucagon positive cells (I, blue cells) are devoid of Pdx1 immunoreactivity. Double- (J, K) or triple-staining (L) of frozen adult mouse pancreas with the monoclonal anti-Pdx1 antibodies F6 (J and L) and F109 (K) with anti-insulin antibody (J, K, L green) and anti-glucagon antibody (L, blue). The monoclonal anti-Pdx1 antibodies recognize islet nuclei in the mature islet β-cells expressing insulin in the cytosol, but not the glucagon producing α-cell. Scale bars: 50 µm except panel l, which is 25 µm.

Rat Pdx1 mRNA expression declines upon exogenous expression of mouse Pdx1. INSrαβ-Pdx1 cells were cultured with/without Dox for 24 hours prior to RNA extraction. Mouse Pdx1 (black) and rat Pdx1 (grey) mRNA expressions were analysed by real-time qPCR and the data was normalised to the housekeeping gene Ppia. The results represent means±sem.