Figure 6.

ADAM10 is responsible for the shedding of PrP^C. (A) Shed PrP^C was immunoprecipitated (IP) from culture supernatants of primary neurons derived from Prnp^0/0, wt, tga20, ADAM10 cKO and littermate control mice (all from E14 embryos) and visualized by Western blot analysis for PrP^C. Shed PrP^C, which shows a 2-3 kDa shift when compared to PrP^C in lysates, is only detectable in wt, tga20, and littermate control neurons. Supernatants from ADAM10 cKO neuron cultures contain virtually no shed PrP^C. IgG light chain (IgG-LC) of capture antibody is detectable at 25 kDa (representative blot is shown). Quantification confirms a significant reduction (p = 0,0026) of the amounts of shed PrP^C shedding in ADAM10 cKO compared to littermate control cultures (Prnp^0/0 values were defined as background and subtracted from the values of all other genotypes; wildtype values were set to one; n = 3 for tga20; n = 5 for A10 cKO and Ctrl). (B) Neural stem (NS) cells derived from an ADAM10 cKO mouse embryo at E14 were transfected (TF) without (+mock) or with Adam10-ORF (+A10). Following neuronal differentiation, PrP^C was immunoprecipitated from culture supernatants (bottom row; IgG-LC = IgG light chain of capture antibody) and detected in corresponding cell lysates (second row; d, m, u = di-, mono-, unglycosylated PrP^C) by Western blotting. Shed PrP^C reappears in Adam10- but not in mock-transfected cultures (bottom row). Success of transfection was verified by Western blot detection of pADAM10 and mADAM10 in cell lysates (top row). Actin is shown as control (third row).