Rabbit anti phospho 4ebp1

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).

Lysates from E13.5 telencephalon were prepared using RIPA buffer and the protein content was determined by a Bio-Rad Protein Assay system. Primary antibodies: rabbit anti-phospho-S6 (Cell Signaling), rabbit anti-phospho-S6K (Cell Signaling), rabbit anti-phospho-4EBP1 (Cell Signaling), rabbit anti-GAPDH (Cell Signaling), rabbit anti-TSC2 (Cell Signaling), rabbit anti-mTOR (Cell Signaling), rabbit anti-phospho-serine (Abcam), mouse anti-GSK3β (BD Transduction Laboratory) or rabbit anti-GSK3β antibody (Upstate). Appropriate secondary antibodies conjugated to HRP were used (Cell Signaling) and the ECL reagents (Amersham) were used for immunodetection.
For quantification of band intensity, blots from three independent experiments for each molecule of interest were used. Signals were measured using ImageJ software and represented by relative intensity versus control. GAPDH was used as an internal control to normalize band intensity.

VLX600 (S8943), DRB18 (S9636), BEZ-235 (S1009) and chloroquine (S6999) were obtained from Selleck Chemicals (Houston, TX, USA). MYCN-amplified neuroblastoma cell lines IMR-32 (Cat. #CCL-127), Sk-N-BE(2) (Cat. #CRL-2271), CHP-212 (Cat. #CRL-2273) and MYCN-nonamplified cell lines SH-SY5Y (Cat. #CRL-2266), Sk-N-AS (Cat. #CRL-2137) were obtained from American Type Culture Collection (ATCC). Primary antibodies used for immunoblotting were the following: mouse anti-MYCN (1:1000, abcam, Cambridge, UK, Cat. #ab16898), rabbit anti-LMO1 (1:1000, Invitrogen, Waltham, MA, USA, Cat. #PA5-77246), rabbit anti-4EBP1 (1:1000, Cell Signaling, Denvers, MA, USA, Cat. #9452), rabbit anti-phospho-4EBP1 (1:1000, Cell Signaling, Cat. #2855), mouse anti-actin (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat. #sc-47778). LC3A/B (1:1000, Cell Signaling Cat. #12741) and MT-COXIV (1:1000, Cell Signaling, Cat. #4850).

Cells were collected and lysed in ice-cold RIPA buffer (with proteinase and phosphor inhibitor) and run on NuPAGE™ 4 to 12% Bis-Tris Mini Protein Gels (Invitrogen) and transferred to nitrocellulose membranes using the iBlot^® 7-Minute Blotting System (Invitrogen). Membranes were pre-treated with SuperSignal^® Western Blot Enhancer Antigen Pretreatment Solution (Thermo Scientific, Waltham, MA, USA) for 10 min, washed according to the manufacturer’s protocol, and subsequently blocked with 5% BSA in 1X PBST for 1 h. The membranes were probed and incubated overnight using the following antibodies in 5% BSA in 1X PBST: mouse anti-MYCN (1:1000, abcam, Cat. #ab16898), rabbit anti-LMO1 (1:1000, Invitrogen, Cat. #PA5-77246), rabbit anti-4EBP1 (1:1000, Cell Signaling, Cat. #9452), rabbit anti-phospho-4EBP1 (1:1000, Cell Signaling, Cat. #2855), mouse anti-actin (1:5000, Santa Cruz Biotechnology, Cat. #sc-47778), LC3A/B (1:1000, Cell Signaling Cat. #12741) and MT-COXIV (1:1000, Cell Signaling, Cat. #4850). The next day, membranes were washed with 1xPBST buffer and incubated with anti-rabbit or anti-mouse secondary antibodies at a 1:5000 dilution for 1 h. SuperSignal^® West Femto Maximum Sensitivity Substrate (Cat. #9452, Thermo Scientific) was used to detect the signal using Amersham Imager 680 (GE Healthcare, Chicago, IL, USA).