(.jpg) Preference for the shedding of fl-PrP over truncated C1 fragment indicates a role of the N-terminal part of PrPC. Western blot analysis of PrP-KO N2a cells transfected with PrP-WT or N-terminally truncated PrP-C1 (corresponding to physiological C1 fragment). Analysis of cell lysates (on the left) reveals N-glycosylation and comparable expression levels for both constructs. Actin served as loading control. Enzymatic deglycosylation (PNGase F) was performed and samples run on a parallel blot to confirm identity of constructs. POM1 antibody was used for detection of PrPC in lysates. Corresponding cell culture supernatants were precipitated and run on a parallel blot (on the right) and shed PrP forms were detected with sPrPG228 antibody. Released sAPPα was detected as loading control for supernatants. Signal intensities of shed PrP forms (sPrPG228 Ab) were referred to total PrP signal intensities in lysates (POM1) and quantification reveals a significantly reduced shedding for PrP-C1 (relative ratio shed/total PrP: 0.12 ± 0.03) compared to (full-length) PrP-WT (set to 1.00 ± 0.11; p = 0.0017; n = 3; ±SEM). (JPEG 799 kb
