Complete protease inhibitor cocktail

CSK buffer was made by dissolving Pipes/KOH (Sigma), NaCl (Sigma), Sucrose (Sigma), EGTA (Sigma) and MgCl₂ (Sigma) in H₂O. Cover slides were coated for 1 h with 20% w/v of fibronectin (Sigma) diluted in PBS (Life Technology) at room temperature. Next, cells were plated on the coated cover slides, washed once with PBS and lysed in CSK buffer supplemented with 0.2% Triton X-100 (Sigma) and cOmplete™ Protease Inhibitor Cocktail (Sigma/Roche) for 5 min at room temperature. Once the incubated solution was removed, the cells were washed with CSK buffer and then were incubated in CSK buffer supplemented with 0.1% Triton X-100 (Sigma), cOmplete™ Protease Inhibitor Cocktail (Sigma/Roche) and 50 U/ml DNaseI (Sigma) for 20 min at room temperature. Cells were then washed with CSK buffer, and the remaining DNA was stained using Hoechst 33342 (Thermo Fisher) at a concentration of 5 μg/ml in CSK buffer supplemented with 125 mM ammonium sulfate (Sigma) and cOmplete™ Protease Inhibitor Cocktail (Sigma/Roche) for 5 min at room temperature. Cells were washed in CSK buffer and fixed in 100% methanol (Sigma) for 5 min at − 20 °C. The methanol was removed and the CSK buffer was added before imaging nucleus acquisition by using DAPI 355 nm on Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). The nucleus area of cells was acquired by a mask function created on the Microscope Software.

Briefly, cells were homogenized in permeabilization buffer (250 mM sucrose, 20 mM HEPES/KOH [pH 7.4], 1 mM EGTA, 1 mM EDTA) containing complete protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany), and centrifuged at 500 g (4 °C) for 10 min to pelletize the nucleus and cell debris. The nuclear pellets were suspended in RIPA lysis buffer (100 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing complete protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany). After vortex and centrifugation at 15,000 g (4 °C) for 15 min, the nuclear supernatants were collected. The supernatants were further centrifuged at 15,000 g for 30 min. Cytosolic supernatants and crude mitochondrial pellets were collected. The pellets were then suspended in mitochondrial lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA) containing complete protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany). After vigorous vortex and centrifugation at 15,000 g (4 °C) for 15 min, the soluble mitochondrial supernatants were collected.

The nuclear extraction was performed according to published procedure [72 (link)]. Cells were harvested with Trypsin-EDTA (0.05%) (Hyclone) and lysed on ice for 5 min with harvest buffer (10 mM HEPES pH 7.9, 50 mM NaCl, 0.5 M Sucrose, 0.1 mM EDTA, 0.5% Triton X-100) supplemented with 1 mM DTT and the Roche complete protease inhibitor cocktail. The lysates were then centrifuged at 1000 g for 10 mins at 4°C. The supernatant was collected as the cytoplasmic part. The pellet was re-suspended with buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA) supplemented with 1 mM DTT and the Roche complete protease inhibitor cocktail and then subjected to another centrifugation at 1000 g for 5 mins at 4°C. The resulting pellet was re-suspended with buffer C (10 mM HEPES pH 7.9, 500 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% IGEPAL (NP40)) supplemented with 1 mM DTT and the Roche complete protease inhibitor cocktail, incubated on ice for 1 h, and then subject to centrifugation at 16000 g for 15 min at 4°C. The resulting supernatant was collected as the nuclear extract of the cells.

Allantoic fluids harvested from eggs were spun at 100,000 × g for 90 min in a SW32 Ti rotor (Beckman Coulter, Brea, CA, USA) into a 20% sucrose cushion to pellet virus. Virus pellets were dissolved in 50 mM HEPES (pH 7), 150 mM NaCl, 1 M urea, 2 mM DTT, 1 tablet of cOmplete Protease Inhibitor Cocktail (Roche) and sonicated briefly to break up aggregates. Dissolved pellets were spun at 5,000 × g for 10 min to remove aggregates loaded onto a 1-ml HiTrap Q HP and a 1-ml HiTrap SP HP column (GE Healthcare Life Sciences), connected in sequence and equilibrated in 50 mM HEPES (pH 7), 150 mM NaCl, 1 M urea, and 1 tablet of cOmplete Protease Inhibitor Cocktail (Roche). After loading, the HiTrap Q HP column was disconnected and M1 eluted from the HiTrap SP HP column using a step gradient of 15% 1 M NaCl. M1 fractions were pooled and stored at −80°C.

Frozen rodent forebrains were homogenized in 9 volumes of ice-cold Tris-buffered saline (pH 7.4) containing Complete protease inhibitor cocktail (Roche Diagnostics, Penzberg, Germany) using a Sonifier 450 (Branson) and stored in aliquots at -80 °C. Triton X-100 soluble Aβ was extracted by mixing 50 μl 2 % Triton X-100 with 50 μl homogenate, incubating for 15 min on ice with vortexing, followed by ultracentrifugation at 100000xg for 15 min. The clear supernatant was diluted to a final forebrain dilution of 1:100 and used for analysis. For the extraction of insoluble amyloid peptides, 50 μl forebrain homogenate was mixed with 117 μl of 100 % formic acid and stored on ice for 15 min with vortexing. Samples were neutralized with 950 μl 1 M Tris base, containing Complete protease inhibitor cocktail (Roche, Basle, Switzerland) and stored overnight at room temperature. The supernatant after 15 min centrifugation at 14000 rpm was used for analysis.

Enriched nuclear proteins samples were prepared essentially as described by Korfali et al [26 (link)], using dissociated testicular cells. In brief, cells were dispersed in 3 ml of ice-cold hypotonic buffer (10 mM HEPES pH 7.4, 1.5 mM MgCl₂, 10 mM KCl, 2 mM DTT) containing Complete protease inhibitor cocktail (Roche) on ice. After 10 min, cells were lysed by application of 20 strokes in a Dounce hand homogenizer. Nuclei were then separated from the cells by a sucrose gradient (0.22 M to 0.9 M). Nuclei were washed in PBS buffer and pelleted at 2000 g at 4°C for 15 min. Nuclei were then solubilized in buffer containing 5 mM MgCl₂, 1% triton X-100, 100 U/ml DNAse and Complete protease inhibitor cocktail (Roche) incubated for 4 hours at 4°C under agitation. After centrifugation at 20 000 g for 30 min at 4°C, the soluble supernatant was conserved and subjected to SDS-PAGE.