N myc

Western blot assays were performed as previously described (24 (link)). Briefly, proteins (20 μg/sample) were separated using SDS–polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes (Bio-Rad). Membranes were probed overnight with the following antibodies: phosphor-AKT (Ser 473), phosphor-AKT (Thr 308), AKT, N-MYC, phosphor-ACC (Ser 79), ACC, phosphor-GSK3b (Ser 9) GAPDH (obtained from Cell Signaling Technology), NAMPT, NAPRT (obtained from GeneTex) or α-tubulin (obtained from Abcam). Membranes were then washed, reacted with horseradish peroxidase–conjugated secondary antibodies and visualized using enhanced chemiluminescence (Thermo Scientific). Relative levels of NAMPT and NAPRT as well as quantitation of cleaved caspase 3 (active) and parp cleavage (indicative of caspase activation) were determined using Image J (imagej.nih.gov).

Cell lysates and tissues were homogenised in RIPA buffer (50 mM TRIS-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 5 mM EDTA) supplemented with a protease and phosphatase inhibitor cocktail (Roche). To terminate the reaction, SDS sample buffer [125 mM Tris- HCl (pH 6.8), 6% SDS, 20% glycerol, and 0.02% bromophenol blue supplemented with 10% β-mercaptoethanol] was added and the samples boiled for 10 min. Proteins were separated on SDS-PAGE 8 to 12% Tris-glycine gels (Life Technologies) and transferred to a nitrocellulose membrane (BioRad). For protein detection, membranes were incubated in 3% BSA in TBS/0.05% Tween-20 blocking buffer for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies from Cell Signaling Technology®: Phospho-FGFR^(Y653/654) (#3471), Phospho-Src Family^(Y416) (#2101), N-Myc (#9405), Phospho-Akt^(T308) (#9275) and GAPDH (#5174), the latter used as a loading control. The following day, membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technologies) at a 1:40000 dilution in 0.75% BSA in TBS/0.05% Tween-20 buffer for 1 h at room temperature. Signal was detected using the chemiluminescent detection reagent Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) and imaged using BioRad ChemiDoc XRS+.