Seeding assay of N2a and GT1 cell cultures with synthetic amyloid fibrils.

<p>Seeding of recMoPrP(23–231) amyloid preparations induced the conversion of endogenous PrP^C to mildly PK resistant forms (A) and accumulation (B) in mouse neuroblastoma N2a and mouse hypothalamic GT1 amyloid-infected cell lines analyzed six passages after the infection (P6). Western blotting shows the partial protease K (PK) resistance of N2a and GT1 amyloid fibril-infected cell lysates. Fibril-infected cell lysates (PK-lanes) were digested with PK at ratio 1:500 (w/w) (PK+ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL) for GT1 infected cells and Clone-P (1μg/mL) for N2a infected cells. Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences) (A). Immunofluorescence imaging shows the accumulations of PrP in N2a and GT1 amyloid fibril-infected cell lines. The deposition and level of PrP (green) in amyloid fibril-infected cell lines after six passages were detected by Fab D18 monoclonal antibody (10 μg/mL final concentration). The nuclei (blue) were stained with DAPI. Scale bar is 20μm (B).</p

PK digestion assay of amyloid #4, #19, #28 and #32.

<p>Western blotting of PK digestion assay (amyloids #4, #19, #28, #32) showed partial protease K (PK) resistance of recMoPrP(23–231) (amyloids #4 and #28). RecMoPrP(23–231) amyloids (PK- lanes) were digested with PK at ratio 1:10 (w/w) (PK+ lanes) and 1:1 (w/w) (PK++ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences)</p

PK digestion assay of GT1 and N2a cells collected at different passages after infection with amyloid fibrils.

<p>Western blotting of GT1 and N2a cell lines infected with PrP amyloid #4 was observed throughout, from first passage (P1) to sixth passage (P6) and after treatment with proteinase K at ratio 1:500 (w/w). Western blot was performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences).</p

Aggregation assay of recMoPrP(23–231).

<p>Recombinant mouse PrP residues 23–231 (recMoPrP(23–231)) was converted <i>in vitro</i> into different amyloid forms (amyloids #4, #19, #28, #32). The amyloid preparations shown (<b>A</b>) exhibited different kinetics for the formation of recMoPrP(23–231) aggregates. Lag phase distribution of amyloid preparations. Bars indicate standard deviation (<b>B</b>).</p

Amplification and characterization of a new prion isolate obtained from the brain of amyloid-infected animals.

<p>PMCA assessment using the brain homogenate of injected animals (with amyloid #4 or #28) as seed and the brain of wild type CD1 animals as substrate. Serial dilution of RML prion strain were used as internal control for PMCA efficiency (A). Western blot (B) and PNGase comparison (C) of amplified clone #4 (PMCA-#4) with known prion strains (RML, mouse adapted vCJD and ME7). Read arrows in B and C indicate the different electrophoretic mobility of the unglycosylated PrP band of PMCA-#4 (migrating at around 20kDa) compared to that of known prion strains migrating at 19 kDa or 21 kDa (black arrows). Western blots were performed using 6D11 monoclonal antibody to PrP (0.2 μg/mL, Covance). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized using a G:BOX Chemi Syngene system.</p

PMCA analysis of raw fibrils and cell lysates from infected N2a and GT1 cell lines.

<p>Seeding ability of amyloid #4 and #28 by means of PMCA using brain homogenates of CD1 mice as substrates for amplification (A). PMCA analysis of GT1 and N2a cell lysates (infected with preparations #4 and #28) and collected at passage six (P6) after the infection (B). Black arrow indicates PK-resistant PrP. Asterisk indicates unspecific binding. Western blots were performed using 6D11 monoclonal antibody to PrP (0.2 μg/mL, Covance). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized using a G:BOX Chemi Syngene System.</p

Western blot analysis of amyloid fibrils performed under reducing and non-reducing SDS-PAGE.

<p>Monomeric (indicated by blue arrow) recMoPrP(23–231) was converted into amyloid forms by intermolecular disulfide linkage following the REDOX process. Western blotting of non-reducing Sodium Dodecyl Sulphate—PolyAcrylamide Gel Electrophoresis (SDS-PAGE) showing the conversion of recMoPrP(23–231) to amyloids is indicated by dimer (green arrow), trimer (red arrow), and more complex structures in both processes (A). Western blotting of reducing SDS-PAGE after treatment of amyloid with Dithiothreitol reducing agent (DTT) shows the decrease in signals of dimer, trimer and more complicated structures in all lanes of amyloid samples from REDOX-process (B). Western blotting of reducing SDS-PAGE of amyloid after a 3-day treatment with denaturant (6M Gdn-HCl), and subsequently with reducing agent DTT shows only monomeric recMoPrP(23–231) bands and the disappearance of more complicated structures in all lanes of amyloid samples in REDOX process (C). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences)</p