Phosphatasearrest

For DNA extraction from monocytes the NucleoSpin® Tissue Kit (Cat No. 740952, Machery-Nagel, Düren, Germany), for RNA extraction the NucleoSpin® miRNA Kit (Cat No. 740971, Macherey-Nagel, Düren, Germany) was applied. Proteins were isolated using RIPA buffer (Cat No. 10017003, Thermo Fisher Scientific, Waltham, Massachusetts, USA), phosphatase arrest (Cat No. 786-782, G Biosciences, St. Louis, USA) and protease arrest (Cat No. 786-108, G Biosciences, St. Louis, USA).

Cell lysates were analyzed by SDS–PAGE and Western blot. Briefly, cells were washed with tris-buffered saline (TBS) and solubilized in ice-cold lysis buffer (50 mM Tris, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1 × ProteaseArrest and 1 × PhosphataseArrest [GBiosciences, St. Louis, MO, USA]) containing 1% SDS. Cells immediately were scraped off the plates and transferred to a microcentrifuge tube. After centrifugation, sample buffer was added to the supernatant in an equal volume, and the entire sample was heated at 100°C for 5 min. Proteins were separated in SDS-PAGE (10% polyacrylamide gels; Bio Rad, Hercules, CA, USA) and transferred onto 0.45 μm nitrocellulose membranes. Membranes were blocked in 5% fat-free milk in TBS (milk), and incubated with primary antibodies overnight at 4°C (1:200 for Alz-50, CP13, and tubulin). An enhanced and optimized 4-chloronaphthal (Opti-4CN, Cat. # 170-8235, Bio Rad) detection procedure was then used. Procedures for the non-infection group were always performed first to avoid accidental contamination.

Cellular protein extraction occurred by resuspending cell pellets in 0.5 mL RIPA buffer (150 mM NaCl, 0.1% Triton X-100, 1% SDS, 50 mM Tris-HCl pH 8) supplemented with PhosphataseArrest (G-Biosciences, Saint-Louis, MO, USA) and protease inhibitors (Complete Mini^®, Roche), Basel, Switzerland). After 15 min incubation on ice with regular vortexing, samples were briefly sonicated (1 min, amplitude 30 kHz, pulse 1 s) and centrifuged at 13,200 rpm for 20 min at 4 °C. Solubilized proteins were transferred to new Eppendorf tubes and stored at −20 °C. Protein lysates were separated using Bis-Tris SDS-PAGE with a high-M_W MOPS running buffer, and transferred onto nitrocellulose membranes (Hybond C, Amersham) using the Power Blotter System (Thermofisher, Waltham, MA, USA). Blocking the membranes for 1 h with blocking buffer (20 mM Tris-HCl, 140 mM NaCl, 5% BSA, pH 7.5) at RT was followed by overnight incubation with the primary antibody at 4 °C. Blots were then incubated for 1 h with the secondary, HRP dye-conjugated antibody (Dako, Glostrup, Denmark) after which chemiluminescent signals were detected with the Amersham Imager 680 (Cytiva, Marlborough, MA, USA) and quantified with the ImageJ software (v1.53j, National Institutes of Health, Bethesda, MD, USA) [94 (link)].