Rabbit anti mtor

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.

SH-SY5Y cells at 60–70% confluency were transfected with the indicated plasmids using Lipofectamine reagent (Thermo Fisher Scientific) as per manufacturer’s instructions. Cells were harvested 48-h post transfection by washing in cold PBS before sonication in RIPA lysis buffer. The lysate was collected after centrifugation at 13,000 rpm, 15 min, 4 °C. 40 μg of protein for each sample was used for electrophoresis (120 V) on 8% SDS-PAGE followed by wet transfer onto nitrocellulose membrane for 80 Volts, 3 h. The primary antibodies used are 1:1000 rabbit anti-PP2A-B (Cell-Signaling Technology, CST), 1:1000 rabbit anti-PP2A-C (CST), 1:2000 guinea pig anti-dS6K (generous gift from Aurelio Teleman), 1:1000 rabbit anti-LRRK2 (Sigma), 1:1000 rabbit anti-phospho-LRRK2 (Ser935) (Abcam), 1:1000 rabbit anti-phospho-LRRK2 (Ser1292) (Abcam), 1:1000 rabbit anti-S6K (CST), 1:1000 mouse anti-phospho-S6K (Thr389) (CST), 1:1000 rabbit anti-mTOR (CST), 1:1000 rabbit anti-phospho-mTOR (Ser2448) (CST) and 1:10,000 mouse anti-actin (Sigma). Detection was performed via chemiluminescence on a Kodak X-ray film developer.

Drug-treated, siRNA-transfected and virus-infected cellular proteins were extracted as follows: cell protein was extracted using IP lysis buffer (Beyotime, Jiangsu, China) and protease inhibitor PMSF (Beyotime). Protein samples with 5 × loading buffer were boiled for 10 min, analyzed by 12% SDS-PAGE, and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were blocked with 3% BSA (Sigma) for 2 h at room temperature, and then incubated with primary antibody for 2 h at room temperature: rabbit anti-LC3B (L7543), rabbit anti-p-mTOR (SAB4504476), mouse anti-β -actin (A1978), rabbit anti-mTOR (SAB2701842), rabbit anti-pTSC2 (SAB4504003), rabbit anti-TSC2 (SAB4503037) (Sigma–Aldrich), rabbit anti-p-AMPK (44-1150G) or rabbit anti-AMPK (AHO1332) antibody (Thermo Scientific). The membranes were incubated with IRDye 800 CW goat anti-mouse IgG or goat anti-rabbit IgG as secondary antibodies (LI-COR Biosciences) for 1 h at room temperature. Detection was carried out using an Odyssey Infrared Fluorescence Scanning Imaging System (LI-COR Biosciences).