Rabbit anti phospho p70s6k

Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies. Proteins were visualized by an ECL detection system (Perbio, Villebon-sur-Yvette, France) using anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (Bio-Rad, Marnes-la-Coquette, France). Densitometry quantitation was determined using the Image J software (NIH, Bethesda, MD, USA).

GSCs were seeded in 100 mm dishes (Falcon) and incubated for 24 hours prior to treatment. After treatment, GSCs were collected and lysed in RIPA buffer, and centrifuged at 12 000 × g for 15 min. Supernatants were collected, and the total protein concentration was quantified using the bicinchoninic acid (BCA) assay kit. Equal amounts of proteins (30 μg) were separated by SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 h, the membranes were incubated with primary antibodies against rabbit anti-active caspase-3, rabbit anti-BAX, rabbit anti-Bcl-2, rabbit antiphospho-AMPK, rabbit anti-AMPK, rabbit antiphospho-acetyl CoA carboxylase (ACC), rabbit anti-ACC, rabbit antiphospho-AKT, rabbit anti-AKT, rabbit antiphospho-mTOR, rabbit anti-mTOR, rabbit antiphospho-4EBP1, rabbit anti-4EBP1, rabbit antiphospho-p70S6K, rabbit anti-p70S6K (All of the above antibodies were procured from Cell Signaling Technology), equal lane loading was confirmed using a monoclonal antibody against β-actin (Sigma-Aldrich). The membranes were washed three times with PBS-T (0.1% (v/v) Triton-X100) buffer for 0.5 hours and incubated with HRP-conjugated secondary antibody for 2 hours. After washing with the PBS-T buffer, the membranes were scanned with the Odyssey Infrared Imaging System (LI-COR).