Fig. 3
From: Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein
Pharmacological manipulation of N-glycosylation alters PrP shedding. a Representative western blot analysis of wild-type N2a cells either left untreated or treated with DMSO (as a solvent control), 2.5 μg/ml tunicamycin (TM) or 5 μg/ml swainsonine (SWA). For TM only unglycosylated PrP is detected. The SWA-treated sample reveals a slight downwards shift for diglycosylated PrP compared to DMSO- and untreated samples. b Endo H digestion further demonstrates an altered glycopattern for SWA-treated cells indicating that correct maturation to complex N-glycans was (at least partially) impaired. Actin served as loading control. c Staining on non-permeabilized cells demonstrating that, in all conditions, PrPC is sufficiently expressed at the plasma membrane. d Representative western blot showing shed PrP in corresponding (precipitated) media supernatants. e Quantification of shedding efficiency (measured as the ratio of shed PrP in supernatants to fl-PrP in respective lysates) showing almost abolished shedding in TM-treated (n = 3; p = 0.002) and impaired shedding in SWA-treated cells (n = 3; p = 0.020)