Preparation of brain homogenates, PK digestion and Western blot analysis

Ten percent (wt/vol) BH of human cases were prepared using 1X LB100 (100 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM EDTA, 100 mM Tris–HCl, pH 8.0), and supernatants (S1) were collected following centrifugation at 1000 × g for 5 min (min) at 4 °C. For the mouse brains, 10% BH prepared in 5% glucose was mixed with an equal volume of 2X LB100 (pH 8.0) and centrifuged at 1000 x g for 5 min. Human and mouse S1 were subjected to enzymatic digestion with 10 Units/ml (U/ml) PK (Sigma Aldrich), which was used at 48 U/mg PK specific activity (1 U/ml is equal to 20.8 μg/ml PK) at 37 °C for 1 h (h). Enzymatic reaction was stopped by the addition of 2 mM phenylmethylsulfonyl fluoride (PMSF) prior to the dilution of each sample with an equal volume of 2 × Laemmli buffer (6% SDS, 20% glycerol, 4 mM EDTA, 5% β –mercaptoethanol, 125 mM Tris–HCl, pH 6.8) and then denaturation at 100 °C for 10 min.

Proteins from human S1 were separated on 15% Tris–HCl SDS–polyacrylamide long gels (W x L: 20 cm × 20 cm) (Bio-Rad PROTEAN® II xi cell system) as originally described [9]. Proteins from the mouse S1 were separated on 15% Criterion™ Tris–HCl Precast Gels (W × L: 13.3 cm × 8.7 cm)[9] (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were blotted onto the Immobilon-FL PVDF membrane (8.7 cm-long gels) or Immobilon-P PVDF membrane (20 cm-long gels) (EMD-Millipore, Billerica, MA, USA), blocked with a blocking buffer and probed with Ab 3F4 (1:10,000), 1E4 (1:500) and Tohoku-2 (1:10,000). The Ab Tohoku-2 was kindly provided by Dr. Tetsuyuki Kitamoto [28]. Membranes were developed by (1) the enhanced chemiluminesce reaction using ECL and ECL plus reagents, and signal was captured on MR and XAR films (20 cm-long gels), or (2) by the Odyssey infrared imaging system (LICOR Biosciences) (8.7 cm-long gels) as described by the manufacturer.