Complete protease inhibitor cocktail tablet

Activity of IDE was measured as reported previously^{68 (link)} using fluorometric SensoLyte® 520 IDE activity assay kit (Cat # AS-72231, AnaSpec, Inc, Fremont, CA, USA) following the manufacturer’s protocol. Briefly, brain frontal cortical tissue extracts were homogenized in cold assay buffer (AnaSpec, Inc, Fremont, CA, USA) supplemented with complete protease inhibitor cocktail tablet (Cat # 11836170001, Sigma Aldrich) plus 0.3 mM PMSF. Homogenates were kept on ice for 30 min, followed by centrifugation at 10,000 X g for 15 min at 4 °C. Protein concentration was measured in the supernatants. Subsequently, enzyme reaction was set up by adding 50 µl of tissue lysates containing 100 µg protein/well in a 96-well black opaque plate. The enzymatic reaction was started by adding 50 µl fluorogenic substrate into each well. The plate was shaken for 30 s, and the reaction was incubated at 37 °C for 60 min in the dark. As a positive control, purified recombinant human IDE provided in the kit was used. The fluorescence intensity was measured at Ex/Em = 490 nm/520 nm using GloMax plate reader (Promega, Madison WI). The fluorescence readings from the wells containing the assay buffer without tissue lysates were used as a background fluorescence. The background reading was subtracted from the reading of the samples, and results are expressed as fluorescence intensity/µg protein.

Cell pellets were resuspended in 50 mM Tris buffer (pH 7.4) containing 100 mM NaCl (Fisher Scientific) and a cOmplete^™ protease inhibitor cocktail tablet (Sigma-Aldrich)). Cells were lysed by incubating them with lysozyme (Sigma-Aldrich) (5 mg/g wet cell paste), DNase (Sigma-Aldrich) (0.1 mg/g wet cell paste), and 2 mM MgCl₂ (Sigma-Aldrich), followed by 10 cycles (each 10 seconds pulse) of sonication with 1 min interval between the successive cycles. First, the soluble components of lysate were removed by centrifugation at 18,000 rpm for 60 min. Then the membranes were washed by resuspending in 10 mM Tris (pH 7.4) buffer containing 100 mM NaCl and protease inhibitors. Finally, upon centrifugation, the washed membranes were used to make the membrane stock solution at 250 mg/mL concentration. All the steps were performed on ice.

All steps were performed at 4°C in the presence of protease inhibitors (cOmplete, protease inhibitor cocktail tablet; Sigma-Aldrich) and phosphatase inhibitors (phosphatase inhibitor cocktail 2 and 3; Sigma-Aldrich). Whole mouse brain or different mouse brain regions were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl) with 20 strokes at 900 rpm. The lysate was incubated under gentle rotation for 15 min, to ensure complete lysis, before centrifugation at 900g for 10 min for removing large cellular debris. Protein concentration was measured by BCA assay. Equal protein amounts were diluted in 1× Laemmli sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting using the corresponding primary antibodies.

For pull-down assays, all steps were performed at 4°C. GST-fusion proteins were expressed in E. coli (BL21) at 16°C for 16 to 18 hours and coupled to Glutathione Sepharose beads (Novagen) according to the manufacturer’s instructions. Whole mouse brain extract was prepared using pull-down lysis buffer (10 mM HEPES and 1% Triton X-100) in the presence of protease inhibitors (cOmplete, protease inhibitor cocktail tablet; Sigma-Aldrich) and phosphatase inhibitors (phosphatase inhibitor cocktail 2 and 3; Sigma-Aldrich). Protein concentration was measured by BCA. GST (30 μg) or GST-fusion protein (30 μg) was incubated with 4 mg of whole mouse brain extract overnight under constant rotation at 4°C. Samples were washed three times using pull-down lysis buffer, boiled with 1× Laemmli sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting using corresponding antibodies.

E. chaffeensis-infected THP-1 cells were harvested at 24, 48 and 72 h post infection (hpi) and whole-cell lysates (uninfected THP-1 cells were used as controls) were extracted three times in complete-RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail tablet (Sigma-Aldrich, St. Louis, MO) and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Lysates were centrifuged at 13,000 x g for 30 s to pellet insoluble material and cleared by centrifugation at 13,000 x g for 20 min at 4°C. The protein concentration was determined using Pierce BCA Protein Assay (Thermo Fisher Scientific). In addition, equal mass amount of N-ethylmaleimide (NEM; Thermo Fisher Scientific) was added to the whole cell lysates to preserve native ubiquitination of proteins in co-immunoprecipitation experiment performed for Fig 4A. Lastly, for the western blot experiment in Fig 4B, 26S proteasome inhibitor, bortezomib (Thermo Fisher Scientific), was added to cell culture at 10ng/ml concentration for 10 h before whole cell lysates were collected.