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Supplementary information
Flow cytometric analysis
Viability of cell cultures was determined by flow cytometric analysis using Fixable Viability Dye eFluor® 780 (eBioscience, San Diego, CA, USA). Flow cytometric analysis of stained cells was performed using a FACSCanto Flow Cytometer equipped with FACSDiva soft- ware (BD Biosciences) and data analysis was performed using FlowJo 7.6 (FlowJo LLC, Ashland, OR, USA).
Western blot analysis
Frozen surgical hippocampal specimens or cells in culture were homogenized in lysis buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, 0.4 mg/ ml Na-orthovanadate, 5 mM EDTA (pH 8.0), 5 mM NaF and protease inhibitors (cock- tail tablets, Roche Diagnostics, Mannheim, Germany). Protein content was determined using the bicinchoninic acid method. For electrophoresis, equal amount of proteins (50 μg/lane) were separated by sodium dodecylsulfate-polyacrylamide gel electrophore- sis (SDS-PAGE, 12% acrylamide). Separated proteins were transferred to nitrocellulose paper by electroblotting for 1 h and 30 min (BioRad, Transblot SD, Hercules, CA). After blocking for 1 h in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.5)/5% non-fat dry milk, blots were incubated overnight at 4°C with the primary antibodies (Table 2; pro- teasomeβ1,1:1000;proteasomeβ1i,1:1000;proteasomeβ5,1:2500; proteasomeβ5i, 1:1000; β-actin, mouse monoclonal, Sigma, St. Louis, MO; 1: 10,000; β-tubulin, mouse monoclonal, Sigma, St. Louis, MO; 1: 2000). After several washes in TBST, the membranes were incubated in TBST / 5% non-fat dry milk, containing the goat anti-rabbit or rabbit anti-mouse antibodies coupled to horse radish peroxidase (1:2500; Dako, Denmark) for 1 h. After washes in TBST, immunoreactivity was visualized using ECL PLUS western blot- ting detection reagent (GE Healthcare Europe, Diegen, Belgium).
Quantification
The images were captured with an Olympus microscope (BX41, Tokyo, Japan) equipped with a digital camera (DFC500, Leica Microsystems-Switzerland Ltd., Heerbrugg, Switzerland). A total set of 6 images from 6 different cases were collected per pathol- ogy. Fiji (ImageJ2) was used for image processing. In a first step colour deconvolution (RGB colour space) was performed in order to separate positive cells from background according to the following channel parameters: red: 0.21408768, green: 0.8171735, blue: 0.4782719. Then a threshold (= 233) was applied and subsequently the images were con- verted to 8 bit gray-scale. The positive pixels/total assessed pixels, indicated as staining percentage area and intensity was calculated.
In situ hybridization
In situ hybridization (ISH) for β1i and β5i was performed using double digoxygenin (DIG) -labeled custom LNA oligonucleotides (Exiqon A/S, Denmark; see supplementary Table 1). The hybridizations were done on 5 μm sections of paraffin embedded materials as pre- viously described 1. The probes were hybridized at 56°C for 1 hour and the hybridization was detected with alkaline phosphatase (AP) labeled anti-DIG (Roche Applied Science, Basel, Switzerland). NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3’-in-