Fig. 3

Immunoblot analysis and immunostaining of human pancreas. a Western blots investigating the presence of proinsulin-immunoreactivity (using antibody GS-9A8, upper panel) and INS-IGF2 fusion protein immunoreactivity (using antibody BO1P, lower panel) in the human beta cell line EndoC-βH1 and HEK, non transduced, GFP transduced or INS-IGF2 transduced. Endogenous proinsulin is marked with (Asterisk) and INS-IGF2 with (filled circle). Notice that INS-IGF2 is only reliably detected in cells transduced with INS-IGF2 construct. We titrated the levels of transduced HEK293 cell extract to give comparable band intensities on EndoC-βH1 proinsulin and HEK293-INS-IGF2 (panel a upper: GS-9A8 which has assumed identical affinities to the two proteins). INS-IGF2 transduction of EndoC-βH1 leads to relative lower expression of INS-IGF2-protein compared to proinsulin (panel a upper comparing the two bands in lane INS-IGF2-EndoC-βH1). This low-level expression of INS-IGF2 protein in transduced EndoC-βH1 is readily detected using the INS-IGF2 antibody while untransduced cells are completely negative (panel a lower). Thus we conclude that the expression of INS-IGF2 protein in EndoC-βH1 is below detection limits of this assay. b Immunoreactivity for INS-IGF2 (green) and glucagon (red) (top panel), and for proinsulin (green) and glucagon (red) (bottom panel) on adjacent sections of human pancreas. Scale bar 100 µM.