Figure 5
ADAM10 is not responsible for α-cleavage of PrPC. (A) Western blot analysis for premature (pADAM10) and mature ADAM10 (mADAM10; upper row), PrPC (second row), PrPC C1 fragment after PNGase F treatment (third row), and actin (bottom row) in neuronal cultures from Prnp0/0, wt, tga20, ADAM10 cKO and littermate controls at E14. Levels of ADAM10 are dramatically reduced in ADAM10 cKO when compared to cultures from littermate controls or from mice overexpressing or lacking PrPC. In accordance with figure 2, PrPC levels are increased in ADAM10 cKO and there is no influence of ADAM10 status on the presence of the C1 fragment, which is most abundant in neuronal lysates with elevated expression of PrPC (i.e. ADAM10 cKO and tga20). (B) Western blot analysis for PrPC in brain of ADAM10 cKO and littermate controls at E14 confirms increased PrPC and the presence of the C1 fragment in ADAM10 cKO mice in an in vivo setting. (C) Western blot analysis for PrPC in lysates of ADAM10 cKO and wildtype neurons (both from E14 mice embryos) confirms increased PrPC amounts in the absence of ADAM10. Soluble N1 fragment was immunoprecipitated (IP) from culture supernatants of these neurons and is found to be increased in supernatants derived from ADAM10 cKO neurons.