Shedding of PrP is decreased in PrP^CGPIThy-1 L150 mice but increases after RML infection.

(A) Representative western blot showing shed PrP (detected with our new antibody described in [36]) and total PrP levels (detected with POM1). The blots for shed PrP and total homogenates were run in parallel (n = 4 for each genotype). The diglycosylated band is preferentially shed in both WTPrPC and PrPCGPIThy-1 L150 brains, but in the latter, shedding is reduced to 27% (***p = 0.0005) as shown in the bar chart (B). To quantify, each blot (shed and total PrP) was first referred to actin prior to the relative quantification of shed PrP referred to total PrP. WTPrPC is set to 100% and error bars are SEM. (C) Representative western blot of shed PrP and total PrP from RML infected brain homogenates of terminally sick mice (n = 4 for WTPrPC and n = 5 for PrPCGPIThy-1 L150). Note that in the infected brains all the isoforms are shed, in both WTPrPC and PrPCGPIThy-1 L150. (D) Bar chart showing the quantification of shed PrP referred to actin. WTPrPC is set to 100%. Note that shedding in PrPCGPIThy-1 L150 is still decreased compared to WTPrPC (***p = 0.0004). (E) Bar chart showing that when shed PrP is referred to the total PrP in RML infected mice, there are no significant differences in the relative amount of shedding, implying a relatively increased shedding in infected PrPCGPIThy-1 L150 brains. Blots were run in parallel and first referred to its actin. WTPrPC is set to 100%. (F) Quantification of the shed PrP glycopattern. Each band intensity was referred to the total amount of shed PrP. In infected brains, the monoglycosylated isoform is significantly less shed in PrPCGPIThy-1 L150 mice (*p = 0.0158).