Fig. 2. PN-1 is internalized via LRP1-mediated endocytosis by all cultured CGNPs. CGNPs from Pn-1 knock-in mice (P8) were cultured for 48 hours on polylysine substrate (500 µg/ml). (A) Pn-1-expressing cells (counterstained by anti-beta-III tubulin in brown) were identified by beta-galactosidase activity (arrowheads). (B) SHH and FGF2 were tested for their ability to stimulate Pn-1 expression in cultured CGNPs from Pn-1 knock-in mice (P5). FGF2 markedly increases Pn-1 expression, whereas SHH does not. (C) LRP1 immunodetection (green) indicates that this receptor is present in/on the cell body and processes of all cells. (D-G) Uptake of recombinant PN-1 is antagonized by blocking LRP1-binding sites. PN-1 immunodetection was performed on CGNPs (P8) incubated for 4 hours with control medium (D), or with medium supplemented with 60 nM PN-1 (E), with 60 nM PN-1 plus 1 µg/ml RAP (F), or with 25 µg/ml P960 or P965 (not shown). Arrowheads show PN-1-containing CGNPs. (G) Quantitation of the mean PN-1 immunolabelling per cell. Values are expressed as mean±s.e.m. (^***P<0.001; Student's t-test). Scale bars: 20 µm in A for A,C; 5 µm in D for D-F.