Fgfr2

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.

Total protein lysates were extracted from untreated cell lines as well as 48 h after siRNA transfection or 2 or 5 h after inhibitor treatment. For western blot analysis equal amounts of total protein were loaded on NuPAGE^® Bis-Tris Gels (Life Technologies). Electrophoretically separated proteins were transferred to nitrocellulose membrane and unspecific binding was blocked with 5% milk/PBS-Tween. Membranes were incubated with primary antibodies overnight at 4°C (FGFR2 1:500; ACTB 1:5000, AC-15, both Sigma Aldrich; FGFR1 1:500; FGFR4 1:500, AM11076PU-N, both from Acris Antibodies, San Diego CA, U.S.A.; FGFR3 1:500, D2G7E; phospho-FGFR 1:500, 55H2; ERK1/2 1:1000; phospho-ERK1/2 1:1000, 20G11, all from CellSignaling Technology, Danvers MA, U.S.A.; HPRT 1:1000, abcam). After incubation with HRP-conjugated secondary antibody SuperSignal West Pico or Femto Chemiluminescent Substrate (Thermo Scientific) was used for detection.