Figure 6
ADAM10 is responsible for the shedding of PrPC. (A) Shed PrPC was immunoprecipitated (IP) from culture supernatants of primary neurons derived from Prnp0/0, wt, tga20, ADAM10 cKO and littermate control mice (all from E14 embryos) and visualized by Western blot analysis for PrPC. Shed PrPC, which shows a 2-3 kDa shift when compared to PrPC in lysates, is only detectable in wt, tga20, and littermate control neurons. Supernatants from ADAM10 cKO neuron cultures contain virtually no shed PrPC. 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 PrPC shedding in ADAM10 cKO compared to littermate control cultures (Prnp0/0 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, PrPC 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 PrPC) by Western blotting. Shed PrPC 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).