Dapk1

After harvesting, the cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1 mM DTT) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail. Mouse and human brain extracts were prepared by homogenizing tissue in ice-cold lysis buffer (10 mM Tris-HCl (pH 7.4), 0.8 M NaCl, 1 mM EGTA, 10% sucrose, 1 mM DTT) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail. The soluble protein concentration was determined by Bradford assay (Bio-Rad). Protein samples (5–10 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (PerkinElmer, Waltham, MA, USA). The membranes were then probed with various antibodies, followed by HRP-conjugated secondary antibodies. Antibody-bound proteins were detected using the Western Lightning Plus-ECL (PerkinElmer) or ECL Prime (GE Healthcare Life Sciences, Pittsburgh, PA, USA) chemiluminescence system. The following antibodies were used to probe different proteins: Flag, DAPK1 (Sigma); DCX, β-actin, α-tubulin (Cell Signaling Technology, Danvers, MA, USA); PHF-13 (p-Ser396-Tau) (Anaspec, Fremont, CA, USA); Tau-5, p-Tau (Ser262); and AT180 (pThr231-Tau) (Invitrogen).

Protein concentration was determined using the Pierce BCA protein assay (Thermo-Fisher). Before undergoing SDS-PAGE, samples were boiled in Laemmli sample buffer for 5 min at 95°C. Proteins were separated in a resolving phase polymerized from 7%–9% acrylamide, then transferred to a polyvinylidene difluoride membrane at 24 V for 1–2 h at 4C. Membranes were blocked in 5% milk or BSA and incubated with anti-CaMKIIa (1:4000, CBa2, available at Invitrogen but made in house), pT286-CaMKII (1:2500, Phospho-Solutions), anti-GluN2B (1:1000, Cell Signaling), DAPK1 (1:800, Sigma-Aldrich), followed by either Amersham ECL goat anti-mouse HRP-linked secondary 1:10000 (GE Healthcare) or goat anti-rabbit horseradish peroxidase conjugate 1:10000 (Bio-Rad). Blots were developed using chemiluminescence (Super Signal West Femto, Thermo-Fisher), imaged using the Chemi-Imager 4400 system (Alpha-Innotech), and analyzed by densitometry (ImageJ). Variations in sample loading were corrected using b-actin (1:2000, Cell Signaling) as a loading control. Relative band intensity was normalized as a percent of control conditions on the same blot, which was set at a value of one to allow for comparison between multiple experiments.