Western blot determination of autophagy markers

Before IHC, the specificity of antibodies against ATG5, LC3-B, LC3-A and p62 was prior determined by Western blot (Supplementary Fig. ^S3). At least 0.5 g of the CNS tissues (Fc, T, Cbl and Mo) were homogenized each in 5 ml of Prionics® Check Western homogenization buffer (Prionics AG, Zurich, Switzerland) and centrifuged twice at 10000 × g for 10 min at 4 °C. The supernatants were analysed by Western blot. Briefly, 25 μg of total protein was subjected to 10% SDS/PAGE and transferred to PVDF membranes (GE Healthcare, UK). After blocking at 4 °C overnight, the membranes were incubated for 1 h at RT with the above described primary antibodies diluted in blocking buffer: anti APG5 (1:1000 dilution); anti MAP-LC3β (1:8000); anti MAP-LC3α (1:1000), and anti p62 (1:1000). In addition, goat polyclonal anti Actin (I-19, sc-1616; Santa Cruz Biotechnology, 1:10000) was used to normalize results. Next, the membranes were incubated for 1 h at RT with a HRP-conjugated secondary antibody diluted 1:4000 in blocking buffer (goat anti-mouse IgG-HRP for APG5 and MAP-LC3β, or goat anti-rabbit IgG-HRP for MAP-LC3α and p62; Santa Cruz Biotechnology, CA, USA). After washing, Western blots were developed using the ECL Plus Western Blotting system (GE Healthcare, UK) and visualized with VersaDoc imaging system (Bio-Rad).

In order to verify the differences observed by IHC for LC3-A and LC3-B and to visualize LC3-I and LC3-II bands, we compared lysates from scrapie (n = 4) and control (n = 4) cerebella. The quantification analysis was performed based on the blotting results using the ImageJ 1.4.3.67 image-analysis software package (Psion Image, NIH) following a simple method of analysis, performed by integrating the grey levels of pixels (volume) surrounded by a rectangular selection. This method is described on the ImageJ website⁶³. Density of immunoreactive bands for autophagy markers was normalized for Actin density band and is reported as arbitrary units (a.u.). Data are expressed as means ± standard error.