Type I interferon protects neurons from prions in in vivo models

Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Mori, Tsuyoshi; Atarashi, Ryuichiro; Nishida, Noriyuki

Immunoblotting was performed as previously described Ishibashi et al., 2015) . The culture cells and animal tissues treated with various experimental conditions were lysed in 1 Â lysis buffer [50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.5% Triton TM X-100, 0.5% sodium deoxycholate, 2 mM EDTA, and protease inhibitors (Nacalai Tesque)], for 30 min at 4 C. The lysates were then treated by sodium dodecyl sulphate (SDS) sample buffer and were separated with 15% SDS-polyacrylamide gel electrophoresis, and blotted to a polyvinylidene difluoride membrane. Bands were detected by appropriate primary antibodies and horseradish peroxidase-labelled secondary antibodies, and visualized using the Chemi-Lumi One L (Nacalai Tesque), ECL prime Western Blotting Detection Kit (GE Healthcare Life Sciences), or Clarity TM Western ECL Substrate (Bio-Rad) to get appropriate results by sufficient enzymatic reaction. Band intensities were quantified using ImageJ software (NIH). For PrP Sc detection, lysates were digested with 20 mg/ ml of proteinase K (Nacalai Tesque) at 37 C for 30 min.