PK-resistant PrP in light fractions of TgMHu2ME199K brain gradients.

<p>Sarkosyl extracted brain homogenates of sick TgMHu2ME199K/wt mice (in the figure: 9 month old score = 4) were subjected to ultracentrifugation in 10–60% sucrose gradients. Individual fractions were digested in the presence or absence of PK and immunoblotted with: (<b>a</b>) α-PrP mAb 6H4 and (<b>b</b>) α-PrP pAb RTC directed against the C-terminal end of PrP.</p

Aggregation of wt and mutant PrP in TgMHu2ME199K/wt mice.

<p>(<b>a</b>) <b>Epitope mapping of α-PrP antibodies:</b> epitopes of antibodies used in this manuscript are depicted on a schematic representation of the chimeric mouse-human E199K PrP. In the next panels (b &c) αPrP mAbs IPC1 and 3F4 were used to differentiate between wt PrP and chimeric-mutant PrP respectively. (<b>b</b>) <b>Oligomeric E199K PrP in asymptomatic TgMHu2ME199K/ko mice:</b> Sarkosyl extracted brain homogenates of wt and 3 months old (score = 0) TgMHu2ME199K/ko mouse were subjected to ultracentrifugation in 10–60% sucrose gradients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069583#pone.0069583-Tzaban1" target="_blank">[34]</a>. Individual fractions were immunoblotted with αPrP mAb IPC1 to detect wt PrP and αPrP mAb 3F4 to detect chimeric-mutant PrP. (<b>c</b>) <b>Oligomeric wt PrP in sick TgMHu2ME199K/wt mice.</b> Sarkosyl extracted brain homogenates from TgMHu2ME199K/wt mice at different ages (1 month, 3 months and 7 months, score = 0, score = 0 score = 3 respectively) were subjected to ultracentrifugation in 10–60% sucrose gradients. Individual fractions of each gradient were immunoblotted with mAb IPC1 to detect wt PrP and mAb 3F4 to detect chimeric-mutant PrP.</p

Correction: PrP^ST, a Soluble, Protease Resistant and Truncated PrP Form Features in the Pathogenesis of a Genetic Prion Disease

<p>Correction: PrP^ST, a Soluble, Protease Resistant and Truncated PrP Form Features in the Pathogenesis of a Genetic Prion Disease</p

PrP forms in soluble and membranal brain fractions.

<p>(<b>a</b>) Fractionation protocol of brain homogenates. (<b>b</b>) Soluble and membranal brain fractions were digested in the presence and absence of PK and immunoblotted with two α-PrP c-terminal Ab; pAb RTC and mAb EP1802Y (EP) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069583#pone-0069583-g002" target="_blank">figure 2a</a> for epitope mapping).</p

Similar disease kinetics and PrP accumulation in heterozygous and homozygous TgMH2ME199K mice.

<p>(<b>a</b>) The percentage of mice presenting a score <2 in both E199K/ko and E199K/wt lines as related to age. Median was 5.2±0.8 for TgMHu2ME199K/ko mice and 6.0±1.2 for TgMHu2ME199K/wt mice. (<b>b</b>) PK-resistant PrP levels are similar in both lines as related to age. Brain homogenates from both TgMHu2ME199K/KO and TgMHu2ME199K/wt lines in different ages and clinical stages (1 month, 3 months and 7 months, score = 0, score = 0 score = 3 respectively) as well as wt and scrapie RML controls were digested with PK and immunoblotted with α-PrP pAb RTC.</p

pAb RVC does not recognize PrP^Sc generated in prion infected cells.

<p>(<b>A</b>) Brain (normal, scrapie infected, as well as scrapie infected digested with PK) as well as normal and prion-infected cells, (N2a and GT1 infected either with the RML or the 22L prion strains) were extracted and immunoblotted with mAb IPC1 as compared to pAb RVC. (<b>B</b>): Effect of the MMA chemical reduction of proteinase K digested ScGT1 and Sc N2a cells on the PrP recognition by mAb IPC1, mAb IPC2 and pAb RVC.</p

Correction: Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc

<p>Correction: Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc</p

HuPrP E200K is spontaneously oxidized.

<p>(<b>A</b>) Brain samples from scrapie infected mice and from humans suffering from familial E200K or sporadic CJD, were digested in the presence or absence of proteinase K and subsequently immunoblotted with mAb 6H4 or pAb RGM. The last 2 lanes of each gel comprise normal GT1 and proteinase K digested ScGT1 cells expressing a chimera Mo/Ha PrP form. (<b>B</b>) Human and mouse normal brain homogenates were immunoblotted with the RGM antibody alone or preincubated with several PrP peptides in the Helix-3 Met area. (<b>C</b>) Immunoblots of HuPrP(23–230) wt and E200K with 3F4 (recognizing the 109–112 region), DZS18 (recognizing oxidized Met residues in different proteins), IPC2 (recognizing non-oxidized M213) and RGM (recognizing non-oxidized M206). Blots were prepared in the absence of β-mercaptoethanol. (D) Thermal stability of HuPrP(23–230) wt and E200K probed by the relative change in the ellipticity at 220 nm as a function of temperature. Insert: Far-UV CD spectrum of HuPrP(23–230) wt and E200K.</p

Intermediate PrP forms are oxidized as PrP^Sc.

<p>Sarkosyl extracted brain samples from normal and prion infected mice and humans were subjected to sucrose gradient centrifugation. Fractions from these gradients were digested in the presence or absence of proteinase K and immunoblotted with both mAb 6H4 and pAb RVC.</p

Scheme of Helix 2 and 3 of PrP including epitopes of α Helix 3 antibodies.

<p>Scheme of Helix 2 and 3 of PrP including epitopes of α Helix 3 antibodies.</p