Genegnome digital imager

Proteins from SDS-PAGE were transferred onto nitrocellulose membranes which were incubated with primary rabbit polyclonal anti-P58 (link), -N58 (link); -M2-126 (link), and mouse monoclonal anti-tubulin-α antibodies (Sigma-Aldrich, Saint-Quentin Fallavier, France) for 1 h, followed by appropriate HRP-conjugated secondary antibodies for 1 h. Immunodetections were performed using an enhanced chemiluminescence (ECL) substrate (GE Healthcare GMB, Vélizy, France). Protein levels were determined from Western blots using the GeneTools software after acquisition of the signals with a GeneGnome Digital Imager (Syngene, Frederick, MD, USA).

Embryos proper, neural tube-enriched samples and 1C11 cells were homogenized in anti-protease (Sigma)-containing buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 300 mM NaCl). The total protein concentration was determined by a bicinchoninic acid assay (Thermo-Scientific). 5–10 μg protein samples were then run on 12% Bis-Tris polyacrylamide gels (Bio-Rad, Marnes-la-Coquette, France), electrotransferred and blotted onto nitrocellulose membranes with the antibodies of interest (Table S2). Immunoreactivity was visualized by enhanced chemiluminescence (Amersham Pharmacia Biosciences, GE Healthcare Europe, Velizy-Villacoublay, France). The protein levels were quantified with the GeneTools software, after acquisition of chemiluminescent signals with a GeneGnome digital imager (Syngene, Frederick, Maryland, United States). The blots were also incubated with anti-β-actin antibody (Table S2) to normalize signals to β-actin as a loading control for quantification.

Brains and spleens were analyzed for proteinase K (PK)-resistant PrP^Sc (PrP^res) content using a previously published protocol [28 (link)]. Briefly, PrP^res was extracted from 20% w/v tissue homogenates with the Bio-Rad TeSeE detection kit. Aliquots were digested with PK (200 μg/mL final concentration) for 10 min at 37°C before B buffer precipitation and centrifugation at 28,000 × g for 15 min. Pellets were resuspended in Laemmli sample buffer, denatured, run on 12% Bis/Tris gels (Bio-Rad), electrotransferred onto nitrocellulose membranes, and probed with 0.1 μg/mL biotinylated anti-PrP monoclonal antibody Sha31 antibody (human PrP epitope 145–152, [72 (link)]) or with 0.1 μg/mL anti-PrP 12B2 antibody (human PrP epitope 89–93, epitope, [42 (link)]) and followed by streptavidin conjugated to horseradish peroxidase (HRP) or by HRP conjugated to goat anti-mouse IgG1 antibody (1/20 000 final dilution), respectively. Immunoreactivity was visualized by chemiluminescence (GE Healthcare). For quantification, the total amount of PrP^res or the relative amounts of PrP^res glycoforms were determined by the use of GeneTools software after acquisition of chemiluminescent signals with a GeneGnome digital imager (Syngene, Frederick, MD).