Cell surface staining of PrP in cerebellar neurons from non-Tg, Tg(Δ23–31^1×), and Tg(Δ23–134) mice.

<p>Cerebellar granular neurons cultured from mice of the indicated genotype were stained for cell surface PrP (6H4, green). Like WT PrP from non-Tg mice, both Δ23–31 and Δ23–134 PrPs are present on the cell surface and along neurites.</p

N-terminally deleted forms of PrP are impaired in their ability to suppress the neurodegenerative phenotype of Tg(F35) mice.

<p>The genotype, number of mice, age at death, and relative expression levels PrP are shown for each transgenic line. While 0.5× expression of WT PrP greatly prolongs the lifespan of Tg(F35) mice, the N-terminal mutants have only a modest effect on lifespan, even at elevated expression levels. Asterisks indicate statistically significant differences in age at death compared to Tg(F35)/<i>Prn-p</i>^−/− mice (<b>**</b> p<0.001, <b>*</b> p<0.01 by Kruskal-Wallis with Dunn's secondary test).</p

Survival of mice co-expressing N-terminal deletion mutants.

<p>Each point represents the percentage of animals alive at the indicated age. Statistical analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025675#pone-0025675-t001" target="_blank">Table 1</a>.</p

F35 does not interact with either WT PrP or Δ23–31 PrP in co-immunoprecipitation experiments.

<p>Brain lysates from mice expressing F35 PrP in the presence or absence of either WT or Δ23–31 PrP were immunoprecipitated with Dynabeads coupled to anti-PrP antibody 6D11, or with naked beads as a control. Immunopreciptated proteins were then analyzed by Western blotting with anti-PrP antibody 6H4. The arrowhead indicates the position of F35 PrP. The faint band appearing in lane 6 between 15 and 20 kDa is distinguishable from the F35 protein, and could represent a C-terminal fragment of Δ23–31 PrP.</p

F35 mice co-expressing N-terminal deletion mutants are normal at 3 weeks.

<p>Animals of the indicated genotypes were sacrificed at 3 weeks and brain sections were stained with hematoxylin and eosin. Images show the whole cerebellum (A–F), the granule cell layer of the second cerebellar lobe (G–L), and the cerebellar white matter (M–R). Scale bars = 1 mm (A–F) and 100 µm (G–R).</p

Expression of transgenes.

<p>(<b>A</b>) Brain lysates from a non-transgenic WT mouse (expressing 1× PrP), and from Tg mice expressing F35 PrP (2×), Δ23–31 PrP (1× and 6×), Δ23–111 PrP (7×), and Δ23–134 PrP (1×) were Western blotted and probed with anti-PrP antibody 6H4. (<b>B</b>) Lysates from the brains of 10 week old mice were treated with PNGase F to removed N-linked oligosaccharides. Digestion products were subjected to Western blotting using antibody 6H4 to detect PrP. Single and double asterisks mark the positions of the endogenous C1 and C2 cleavage fragments, respectively.</p

Δ23–31, Δ23–111, Δ23–134, and Δ32–134 (F35) PrP are GPI-anchored and have a cellular localization pattern similar to WT PrP.

<p>(<b>A–L</b>) The indicated constructs were transiently expressed in BHK cells along with dsRedER. Cells were incubated in the absence (A–F) or presence (G–L) of PIPLC, then surface stained for PrP (A–C, G–I: 6D11 or D–F, J–L: 6H4) on ice prior to incubating with secondary antibody (dsRedER signal in red, PrP in green). DAPI staining is shown in blue for panels D–F, J–L. Like WT PrP (H), the mutant PrP molecules are released from the plasma membrane by PIPLC treatment (I–L). (<b>M–R</b>) BHK cells transfected with the indicated constructs were permeabilized and stained with anti-PrP antibody [M–O: 6D11, P–R: 6H4 (green)], anti-giantin antibody (red), and DAPI (blue). Like WT PrP, each mutant is present both at the cell surface and intracellularly, where it colocalizes with the Golgi marker, giantin. [Scale bar in A (applicable to panels A–L, P–R) = 25 µm. Scale bar in M–O = 15 µm.].</p

Surface plasmon resonance shows that monoclonal antibody 15B3 selectively captures PG14 PrP aggregates from P3 fractions.

<p>Fractions P3 prepared from whole brains of Tg(PG14) or <i>Prnp</i>^0/0 mice were perfused for 5 min (bars) over the sensor surface of SPR chips on which antibody 15B3 (A) or 3F4 (B) had been immobilized. The sensorgrams (time course of the SPR signal in Resonance Units, RU) refer to the specific binding to the antibody (binding was negligible on sensor surfaces without antibody). Significant binding was detected when P3 fractions containing PG14 PrP (black line) were flowed over the 15B3-coated chip (A), but not on the 3F4-coated chip (B). No binding was observed when a P3 fraction from <i>Prnp</i>^0/0 mice was analyzed (gray line).</p

Diagram of the 15B3-immunopurification procedure.

<p>The scheme summarizes the main steps of the purification protocol (A), and illustrates the putative mechanism by which sonication elutes aggregated PrP from 15B3-coated beads (B).</p

Immunopurified aggregates of PrP^Sc support <i>in vitro</i> misfolding of PrP^C by PMCA.

<p>Aggregates of PrP^Sc immunopurified from P3 fractions of RML-infected C57BL/6 mice (lanes 1–8), or immunoprecipitates of P3 fractions of <i>Prnp</i>^0/0 mice (lanes 9–12) were resuspended in PBS (lanes 1–4 and 9–12) or immobilized on glass coverslips by ultracentrifugation (lanes 5–8), and mixed with brain homogenates of Tg(WT) mice overexpressing 3F4-tagged PrP^C. Mixtures were either rapidly frozen (lanes 1 and 2, 5 and 6, 9 and 10) or subjected to 90 cycles of sonication/incubation (lanes 3 and 4, 7 and 8, 11 and 12). Samples were then either subjected to proteinase K (PK) (lanes 2, 4, 6, 8, 10 and 12), or left undigested (lanes 1, 3, 5, 7, 9 and 11). All samples were analyzed by Western blotting with 3F4 antibody.</p