Phosphatase inhibitor

H2452 cells were cultured at a density of 5 × 10⁵ cells in a 60 mm dish for 24 h and then treated with each agent (α-T3E, bortezomib, and marizomib) for 12–24 h. After the treatment, cells were harvested and lysed in ice-cold Laemmli sample buffer (Bio-Rad, Berkeley, CA, USA) containing protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan) and phosphatase inhibitor (Nacalai Tesque). Cells were incubated on ice for 20 min following centrifugation at 12,000 rpm at 4 °C for 10 min. The samples were electrophoresed through a 10% or 15% SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were blocked with Blocking One P (Nacalai Tesque) for 1 h, incubated with primary antibodies for 1 h, and then incubated with the secondary antibody for 1 h. Detection was accomplished using Chemi-Lumi One Super (Nacalai Tesque) and C-DiGit (LI-COR, Lincoln, NE, USA). A densiometric analysis of each immune band was performed using Image Studio for C-DiGit (LI-COR). Molecular sizing was conducted using Rainbow MW markers (Amersham Japan, Tokyo, Japan). Protein concentrations were assessed using the DC Protein Assay System (Bio-Rad).

Tissues were homogenized in ice-cold RIPA buffer (50mM Tris-HCl pH 7.6, 0.15M NaCl, and 1% NP-40) containing cocktails of phosphatase inhibitor (Nacalai Tesque, Kyoto, Japan) and protease inhibitor (Sigma-Aldrich, St Louis, MO, USA). After centrifugation at 800 × g for 20 min at 4°C, the middle layer was taken as total protein to avoid taking the upper fat layer and used for western blotting analysis. In brief, the protein was separated by SDS-PAGE and transferred to a polyvinylidine fluoride membrane (Immobilon; Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk (Morinaga Milk Industry Co., Tokyo, Japan), the membrane was incubated with a primary antibody overnight at 4°C. Primary antibodies against UCP1 (Abcam, Cambridge, UK), COX4 (Thermo Fisher Scientific), and Tubulin (Sigma, St. Louis, USA) were used. The bound antibody was visualized using a horseradish peroxidase-linked secondary antibody and an enhanced chemiluminescence system (GE Healthcare UK Ltd., Little Chalfont, Bucks, UK).

Adherent cells cultured for 24 to 48 h were harvested on ice with a cell scraper. An additional culture of serum-starved cells was trypsinized and kept in suspension for 1 h, then collected in tubes. The cells were the incubated at 37°C for 15 and 30 min, and harvested (Cheng et al., 2014 (link)). The cell suspensions were centrifuged at 300 ×g at 4°C for 5 min and the supernatant was discarded. The cell pellets were washed with chilled phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay lysis buffer (Nacalai Tesque, Kyoto, Japan) or NP40 lysis buffer (Wako, Osaka, Japan) containing protease inhibitor (Nacalai Tesque) and phosphatase inhibitor (Nacalai Tesque) cocktails for 5 min on ice. The cell lysate was centrifuged (16000 ×g at 4°C for 15 min). The lysate [10–20 µg protein, as measured with a BCA Protein Assay kit (Thermo Fisher Scientific)] was then mixed with SDS sample buffer (Nacalai Tesque), separated by SDS-PAGE using pre-made 7.5% or 5–20% polyacrylamide gel plates (e-PAGEL, Atto, Tokyo, Japan), transferred to iBlot® 2 Transfer Stacks PVDF mini membranes using an iBlot® 2 Dry Blotting system (Thermo Fisher Scientific), and immunoblotted with specific antibodies at 1:1000 to 1:5000 dilution (Alanko et al., 2015 (link); Torisu et al., 2013 (link)).

NK-4 (5 μM)-treated THP-1 macrophages were incubated with or without Apo-J cells at a ratio of 1:1 for 45 min at 37 °C. Whole-cell extracts were prepared with RIPA buffer (Wako Pure Chemical) containing phosphatase inhibitor (Nacalai Tesque Inc., Kyoto, Japan) and protease inhibitor (Roche Diagnostics, Mannheim, Germany) and subjected to western immunoblotting. Membranes were probed with a 1:1000 dilution of anti-phospho-Akt (Ser473) rabbit pAb (9171; Cell Signaling Technology, Danvers, MA). Specific bands were detected using an ECL™ Plus Western Blotting System (Immobilon Western Chemiluminescent HRP substrate; GE Healthcare, UK). After treatment with a reprobing solution (Restore Western Blot Stripping Buffer; Pierce Biotechnology, Rockford, IL) for 15 min at room temperature, the membrane was used for secondary detection with a 1:1000 dilution of anti-Akt rabbit mAb (C67E7; Cell Signaling Technology). Band density was measured using ImageQuant TL software (GE Healthcare).

Protein extraction from cultured iPSCs and neurons was performed using a mixture of Complete Mini (Sigma-Aldrich), phosphatase inhibitor (Nacalai Tesque) and Tissue Extraction Reagent I (Invitrogen) consisting of 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, 2 mM Na₃VO₄, 1 mM NaF, 20 mM Na₄P₂O₇, and 0.02% NaN₃, which was added to each well on ice. The samples were collected in tubes, vortexed for 2 s, and then sonicated for 20 s. Centrifugation was performed at 15,000 rpm for 5 min at 4 °C, and the lysates were stored at − 80 °C. The samples were diluted with equal volumes of Laemmli sample buffer (Bio-Rad) containing 62.5 mM Tris–HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% bromophenol blue, and 5% 2-mercaptoethanol and then incubated for 3 min at 95 °C. Protein samples were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 1% nonfat skim milk for 30 min at room temperature (RT) and incubated in primary antibody for 16 h at 4 °C. Thereafter, the membranes were incubated with secondary antibodies for 2 h at RT. Signals were detected using Chemiluminescence HRP Substrate (Takara Bio) with a Lumino Graph I system (ATTO). Semiquantitative analysis of the signals was performed using ImageJ software (National Institute of Health).

Postsynaptic density (PSD) fractions were prepared as described previously (Tada et al., 2016). Dounce homogenate was prepared from the hippocampus and centrifuged at 1,000 g for 10 min to remove nuclei and debris (P1). The supernatant was spun at 12,000 g for 20 min to obtain a P2 fraction. P1 and P2 fractions were resuspended and centrifuged twice to remove contaminants. The P2 fraction was then resuspended in buffer containing 0.5% Triton X‐100 and rotated for 15 min. This fraction was then centrifuged at 100,000 g for 60 min to yield soluble and insoluble fractions (PSD fraction), and the insoluble fraction was then solubilized into T‐PER (Thermo Fisher Scientific, USA). All fractionation steps were performed at 4°C in the presence of 0.32 M sucrose and 4 mM Hepes, containing phosphatase inhibitor (Nacalai tesque, Japan) and complete protease inhibitor mixture (Roche, Swiss).