Fig. 1.

A neural progenitor cell model of FENIB. A. Real-time RT-PCR quantification of NS gene expression in NPCs cultured in proliferating conditions, using primer pairs targeting both the exogenous human NS transgene and the endogenous mouse NS gene. Results are shown as the mean +/− SEM of the ratio between NS and GFP samples of three independent repeats. B. Cell lysates and culture media of stably transfected NPC cultured in proliferative conditions expressing a control protein (GFP), WT, G392E or delta NS (dNS, black arrow, treated or not with the reversible proteasomal inhibitor MG132 at 2 μM for 12 h) were resolved by 10% w/v acrylamide SDS (top and middle panels) or 7,5% w/v acrylamide non-denaturing (bottom panel) PAGE, followed by western blot. Arrowheads: WT NS monomer; vertical line: NS polymers. C. Quantification of intracellular total NS and NS polymers in proliferative NPCs by sandwich ELISA using monoclonal antibodies 1A10 + 10B8 (left graph) or 7C6 (right graph) respectively. The values correspond to NS normalised to total protein and are the mean +/− SEM of three independent repeats. D. Immunofluorescence analysis of WT, G392E and delta NS, as well as negative control GFP proliferative NPCs, with a rabbit anti-NS polyclonal antibody (upper and middle panels) and with monoclonal antibody 7C6 for detection of NS polymers (lower panels, arrow). E. Immunofluorescence colocalisation of WT and G392E NS with resident proteins of the secretory pathway in proliferative NPCs. Cells were costained with a rabbit anti-NS polyclonal antibody and antibodies against KDEL (ER) and GM130 (Golgi apparatus). The colour corresponding to each antibody is indicated for each set of panels, and only the merged images are shown. The nucleus appears blue due to DNA staining with DAPI. Scale bar: 10 μm.