Lysine residues are important for the pre-OR residues 23–31 to form a PK-resistant structure in prion-infected N2a cells.

<p>(A) Amino acid sequences of the pre-OR residues 23–31 in moPrP(3F4) Δ32–88, moPrP(3F4) Δ32–88(3K3A) and moPrP(3F4) Δ32–88(2P2A). Bold residues indicate substituted alanine residues. (B) Western blotting of N2aC24L1-3 cells transfected with control pcDNA3.1(+) and expression vectors encoding moPrP(3F4), moPrP(3F4) Δ32–88, moPrP(3F4) Δ32–88(3K3A) and moPrP(3F4) Δ32–88(2P2A) using 3F4 anti-PrP antibodies. The cell lysates were treated with PK at 5 µg/ml. All mutant proteins were converted into PK-resistant isoforms in N2aC24L1-3 cells. The PK treatment revealed doublet non-glycosylated and mono-glycosylated bands in moPrP(3F4)^ScΔ32–88 (arrows), indicating that the pre-OR region of some moPrP(3F4)^ScΔ32–88 molecules is PK-resistant. Similar doublet bands were observed in moPrP(3F4)^ScΔ32–88(2P2A) (arrows). However, moPrP(3F4)^ScΔ32–88(3K3A) gave rise to doublet bands with the upper band migrating very closely to the lower band (arrowheads). (C) Since substitution of proline residues into alanine residues disrupted the IBL-N epitope, the PK-resistant pre-OR residues in moPrP(3F4)^ScΔ32–88(2P2A) failed to be visualized by IBL-N anti-PrP antibodies.