Complete protease inhibitor cocktail and phosstop

Frozen samples of the heart, liver and white adipose tissue (WAT) were obtained from 6 animals in each group. The samples were placed in 4 volumes of ice-cold TMES buffer (20 mM Tris, 3 mM MgCl₂, 1 mM EDTA and 0.25 M sucrose; pH 7.4) containing protease and phosphatase inhibitors (cOmplete Protease Inhibitor Cocktail and PhosSTOP; Roche Diagnostics, Basel, Switzerland), cut into small pieces, and homogenized in an ice-bath with a Potter-Elvehjem homogenizer (20 strokes at 1200 rpm) on ice. The resulting suspension was centrifuged at 2000× g for 10 min (4 °C) to remove large tissue debris and nuclei. The resulting post-nuclear supernatant (PNS) was centrifuged at 33,000× g for 20 min (4 °C) to isolate crude plasma membranes (CPM). The pelleted membranes were re-suspended in half of the initial volume of TMES buffer containing 1% Nonidet P-40. The supernatant was centrifuged at 200,000× g for 60 min to isolate the light microsomal fraction (LM). The pellet was then re-suspended in half of the initial volume of TMES buffer containing 1% Nonidet P-40. Aliquots of CPM and LM were snap-frozen and stored at −80 °C.

HEK293T cells were seeded in petri dishes, using antibiotic-free DMEM medium, to 70% confluency and then transfected with the plasmids outlined in the figures using Lipofectamine® LTX (Invitrogen) according to the manufacturer’s recommendations. For individual transfections, 8 µg of plasmid DNA was used, whereas for the co-transfection, 4 µg of each plasmid was utilized. The final volume after adding the transfection mixtures was 2 mL. After 6 h incubation, cells were detached, washed in PBS, and centrifuged. Pelleted cells were resuspended in 500 µL lysis buffer (30 mM Tris-HCl pH7.5, 120 mM NaCl, 1% glycerol, 0.5% NP-40, supplemented with Complete Protease Inhibitor Cocktail and PhosSTOP (Roche Diagnostics)), and subsequently left on ice for 30 min, followed by centrifugation at 13,684g for 20 min. The lysates were then incubated with either anti-RFXANK- or anti-RFP-coated Dynabeads Protein G (Thermo Scientific) overnight at 4 °C, under constant rotation. Each sample was then placed in a DynaMag2 (Life Sciences), allowing the bead complexes to be separated from the unbound proteins (i.e., flow-through). Subsequently, each sample was washed with lysis buffer for 3 × 5 min under rotation at room temperature. Finally, each complex was resuspended in 30 µL 1x Laemmli buffer and analyzed by Western blotting.

Human brain tissue from the frontal cortex from individuals with AD and controls was sonicated twice for 20 s in 10 × w/v RIPA buffer plus protease and phosphatase inhibitors (Roche, Basel, Switzerland), in a cold room. Homogenates were centrifuged for 10,000 rpm at 4°C for 10 min and the supernatant was collected for further analysis. Mouse hemi-brains were sonicated twice for 10 s each in a cold room in 10 × w/v buffer consisting of 10 mM Tris/HCl pH 7.5, 0.8 mM NaCl, 1 mM NaF, 1 mM Na3VO4, complete Protease Inhibitor Cocktail and PhosSTOP (Roche, Basel, Switzerland), and 10 μM anacardiac acid (Sigma-Aldrich, St. Louis, MO, USA). This homogenate was termed crude homogenate (CH). CH was divided for Western Blot and Mass Spectrometry analysis.