T per protein extraction buffer

Mouse proteins were prepared as previously described (Velazquez, Shaw, Caccamo & Oddo, 2016; Velazquez et al., 2017). One hemisphere of the brain was postfixed in 4% paraformaldehyde for 48 hr while the other hemisphere had the hippocampus and cortex isolated, flash‐frozen in dry ice, and stored at −80°C. A subset of hemispheres were dropped in Golgi–Cox solution following the manufacturer protocol (Rapid Golgi; FD NeuroTechnologies). The frozen brain regions were homogenized in ice‐cold T‐PER protein extraction buffer (Thermo Fisher Scientific) containing complete protease inhibitor (Roche Applied Science) and phosphatase inhibitor (Life Technologies). The homogenized mixtures were centrifuged at 100,000 g for 1 hr at 4°C. The resulting supernatant was recovered and stored at −80°C and used for Western blots, which were performed under reducing conditions using precast Novex gels (Life Technologies). Quantitative analyses of the Western blots were obtained by normalizing the intensity of the protein of interest with its loading control, β‐actin.

Human and mouse proteins were prepared as previously described [10 (link)]. Briefly, mice were sacrificed and their brains were removed and cut in half along the medial longitudinal/sagittal fissure. One hemisphere of the brain was post-fixed in 4 % paraformaldehyde for 48 h and used for immunohistochemical evaluation. The other hemisphere was flash-frozen on dry ice and used for biochemical experiments and stored at −80 °C. The frozen mouse hemispheres as well as 0.1 g of human inferior frontal gyrus tissue were mechanically homogenized in ice-cold T-PER protein extraction buffer (Thermo Fisher Scientific) containing complete protease inhibitor (Roche) and phosphatase inhibitor (Life Technologies). Brain homogenates were ultracentrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was recovered and stored at −80 °C until used for western blots and for both pPRAS40 and soluble Aβ levels by ELISA. The pellet was re-suspended in 70 % formic acid, mechanically homogenized, and centrifuged as described above. The supernatant of this second centrifugation was recovered and stored at −80 °C until used as the insoluble fraction for ELISA experiments.