See supplementary figure 1 for binding curves.
Figure 5. Binding of the two radioligands to rodent GLP-2Rs and autoradiography . (a, b) Competition binding of [125I]-hGLP-2(1-33,M10Y) (black) and [125I]-hGLP-2(3-33,M10Y) (red) to (a) the mGLP-2R(n = 3) and (b) the rGLP-2R (n = 3) displaced by increasing concentrations of endogenous hGLP-2(1-33). To compensate for inter-assay variations the data were normalized to the specific binding of respectively mGLP-2R and rGLP-2R for each individual radioligand within each assay. The experiments were carried out in duplicated and presented as mean ± SEM. Histological sections of the small intestine (c, e) and endocrine pancreas (d, f) after autoradiography in mice injected with (c, d) [125I]-hGLP-2(1-33,M10Y), and (e, f) [125I]-hGLP-2(1-33,M10Y) plus excess amount of unlabeled hGLP-2(1-33,M10Y). The histological sections were counterstained with hematoxylin. Scale bar 50µm.
Figure 6. cAMP accumulation mediated by hGLP-2 variants at the hGLP-1R. cAMP accumulation for the hGLP-1R stimulated with increased concentration of hGLP-1 (n = 9) and, (a) hGLP-2(1-33) (n = 7) and hGLP-2(1-33,M10Y) (n = 3) , (b) hGLP-2(3-33) (n = 6) and hGLP-2(3-33,M10Y) (n = 6) , (c) hGLP-2(1-33) in the presence of 100nM exendin(9-39) (n = 5) , and (d) hGLP-1 in the presence of 1µM hGLP-2(3-33,M10Y) and 100nM hGLP-2(3-33,M10Y) (n = 3) for the hGLP-1R. To compensate for inter-assay variations data have been normalized to hGLP-1 within each separate experiment. The experiments were carried out in duplicated and presented as mean ± SEM.
Supplementary figure 1. Test for selectivity among class B1 GPCRs. (a-f) Competition binding curves of [125I]-hGLP-2(1-33,M10Y) (black) and [125I]-hGLP-2(3-33,M10Y) (red) to (a) hGLP-1R(n = 3) , (b) hGIPR (n = 2) , (c) hGCGR (n = 2) , (d) hSecretinR (n = 2) , (e) VPAC-1R (n = 2) , and (f) VPAC-2R(n = 2) displaced by increasing concentrations of endogenous hGLP-2(1-33). To compensate for inter-assay variations data have been normalized for each individual radioligand within each assay. The experiments were carried out in duplicated and presented as mean ± SEM.
Supplementary figure 2. Autoradiography in mice using [125I]-hGLP-2(1-33,M10Y). Histological sections of the small intestine (a-f) and pancreatic islet cells (g-l) after autoradiography in mice, injected with [125I]-hGLP-2(1-33,M10Y) (a-c and g-i) or [125I]-hGLP-2(1-33,M10Y) plus excess amount of unlabeled hGLP-2(1-33,M10Y) (d-f and j-l). The histological sections were counterstained with hematoxylin. Scale bar 50µm.
Supplementary figure 3. Binding of the two hGLP-2 radioligands to rodent GLP-1Rs. (a, b) Competition binding of [125I]-hGLP-2(1-33,M10Y) (black) and [125I]-hGLP-2(3-33,M10Y) (red) to (a) the mGLP-1R(n = 3) and (b) the rGLP-1R (n = 3) displaced by increasing concentrations of endogenous hGLP-2(1-33). To compensate for inter-assay variations the data were normalized to the specific binding of, respectively, mGLP-1R and rGLP-1R for each individual radioligand. The experiments were carried out in duplicated and presented as mean ± SEM.