Rabbit anti phospho p70 s6

Total brain tissues were sonicated using a LabSonic homogenizer (B. Braun Biotech Inc., Allentown, PA, USA), and the protein concentration in the brain samples was then quantified using a bicinchoninic acid assay kit (Pierce, CA, USA). Samples were then analyzed by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% milk containing 0.05% Tween in PBS. The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel). Membranes were then probed with secondary horseradish peroxidase-conjugated antibodies (1:3000; Amersham Biosciences, Little Chalfont, UK) for 1 h at room temperature. The blots were then visualized using chemiluminescent peroxidase substrate (ECL prime; GE Healthcare). Immunoreactivity for each protein band intensity was quantified using NIH ImageJ software [24 (link)] and normalized to β-actin as a loading control.