Figure 4. Search for a defined domain within PrP^C that regulates its α-cleavage.

A: Amino acid sequence alignment of the PrP^C constructs used. Numbers represent amino acid residues. The positive residues, the charge cluster, hydrophobic core, palindrome and the conserved 100–104 domain are highlighted. Arrow: α-cleavage site. B: Western blot of PNGase treated cell lysates of Hpl cells transfected with the PrP^C deletion constructs used in the current study. Detection was done with POM1. Arrows point to the full length PrP (FL) and C1 fragment. C: Quantification of the percentage of α-cleavage of the various PrP^C mutants, based on densitometry of western blots, from which (B) is representative. Quantifications were calculated in the linear range of the densitometric signal of independent experiments. Values refer to the amount of C1 generation comparing to total abundance of PrP^C, and are normalized to cleavage of PrP^C, which was assessed in the same blot. Error bars represent the SEM. D: Graphic illustrating the percentage of α-cleavage impairment of PrP^C deletion mutants. Points correspond to the average value illustrated in (C), for the samples PrP^C, BOM16, BOM15, BOM17 and C40, which have 0, 4, 9, 14 and 30 amino acids deleted, respectively. In x-axis are plotted the size of the deletion of each of these constructs. Dashed line refers to the estimated deletion size that would result in 50% inhibition of α-cleavage.