Phospho fgfr y653 654

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+.

Primary antibodies used for immunoblotting were as follows: total and cleaved forms of PARP and caspase-3, phospho-KIT Y719, phospho-AKT S473, AKT, ABCG2, phospho-FGFR Y653/654 (Cell Signaling, Danvers, MA, USA), FAK, phospho-FAK Y397 (BD Biosciences, Franklin Lakes, NJ, USA), KIT (DakoCytomation), phospho-Met Tyr1230/1234/1235 (R&D Systems, Minneapolis, MN, USA), Axl, FGFR2 (Sigma), c-Met, MDR, MRP-1 and actin (Santa Cruz Biotechnology, Dallas, TX, USA). The HRP-conjugated secondary antibodies for Western blotting were purchased from Santa Cruz Biotechnology.

Primary antibodies used for immunoblotting and immunofluorescence were as follows: phospho-MAPK (Erk1/2) Thr202/Tyr204 (#4370S), MAPK (Erk1/2) (#4696S), phospho-AKT S473 (#4060P), AKT (#4691p), phospho-FGFR Y653/654 (#3476S), FGFR1 (#9740S), FGFR2 (#23328S) phospho-KIT Y719 (#3391S) (Cell Signaling, Danvers, MA, USA), c-KIT (#A4502, Dako, Carpinteria, CA, USA), beta-actin (A00730-200, GenScript, Piscataway, NJ, USA). HRP-conjugated secondary antibodies used for Western blotting were from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies used for IHC-staining were as follows: c-KIT (#14-1172-82, Thermo Fisher Scientific, Waltham, MA, USA), DOG-1 (#244R, Cell Marque, Rocklin, CA, USA), a cleaved form of caspase-3 (#9662S, Cell Signaling, Danvers, MA, USA) and FGF-2 (sc-365106, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Neutralizing antibody against bFGF (Anti-FGF2/basic FGF #05-117) and human recombinant FGF-2/basic (FGF2 #01-106) were obtained from Merck KGaA (Darmstadt, Germany).