Bicinchoninic acid analysis

Platelets, megakaryocytes, HepG2 cells and HL‐60 cells were lysed in RIPA buffer. After determination of the protein concentration by bicinchoninic acid analysis (Thermo Scientific), 18 μg of total protein was mixed with 100 mm dithiothreitol and Nupage loading buffer (Invitrogen), and boiled for 5 min. The presence of complement proteins was tested for with reducing SDS‐PAGE on 6–7% polyacrylamide gels under standard conditions. Proteins were transferred to nitrocellulose membranes, which were blocked with 5% skimmed milk in PBS containing 0.1% Tween‐20 prior to an overnight incubation with C3c–horseradish peroxidase (HRP) sheep polyclonal antibody (1 : 1000; Life Span Biosciences, Seattle, WA, USA) or C3d rabbit polyclonal antibody (1 : 3000; Dako). Goat anti‐rabbit H + L HRP (1 : 50 000; BioRad, Hercules, CA, USA) was used as the secondary antibody, and the signal was developed with the ECL West Pico and West Femto detection system (Thermo Scientific).

Brain tissue or cell pellets were lysed and homogenized in RIPA buffer containing protease and phosphatase inhibitors. Homogenates were centrifuged at 10,000 g for 15 minutes at 4°C and supernatant was collected. Bicinchoninic acid analysis (Thermo Fisher Scientific) was used to determine and normalize protein concentrations. Protein separation was performed by electrophoresis using 10–15% SDS-polyacrylamide gels. Following separation, proteins were transferred to a nitrocellulose membrane. Nonspecific binding was blocked by 5% BSA in tris-buffered saline then primary antibodies were incubated overnight at 4°C. Primary antibodies were used at the following concentrations; PPARα (1:1000; Cell Signaling), APP (1:1000; recognizes APP-FL and CTF, Cell Signaling), β-actin (1:10,000; Sigma-Aldrich), pS6k (1:1000; Cell Signaling), total S6k (1:1000, Cell Signaling), pAKT (1:1000, Cell Signaling), total AKT (1:1000, Cell Signaling) and γ tubulin (1:1000, Cell Signaling). After primary antibody incubations, secondary antibodies; IR-680-conjugated goat anti-mouse or goat anti-rabbit (1:10,000; Molecular Probes) and IRDye 800-conjugated donkey anti-rabbit or donkey anti-mouse (1:10,000; LI-COR) were used. LI-COR Odyssey machine (LI-COR) was used to image the membranes. The Western blot bands were quantified using ImageJ software (NIH).