Sc 123

The cells were homogenized in lysis buffer (50mM Tris/HCl pH 7.4, 500mM NaCl, 1% NP-40, 0.5% Na-DOC, 0.1% SDS) supplemented with 20μg/ml complete protease inhibitor cocktail (Roche, Mannheim, Germany), 5mM NaF and 100μM Na-vanadate. Aliquots containing 20μg of protein were analyzed by electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene-difluoride membranes. Proteins were identified using antibodies to phospho-FRS2α (Cell signalling, Boston, MA; #3861), phospho-FGFR (Cell Signalling; #3471) and FGFR3 (#sc-123, recognizes both FGFR3 splice variants; Santa Cruz Biotechnology, Inc., Dallas, TX); ERK1/2 (Upstate, Lake Placid, NY #06-182; 1:5000), phospho-ERK (Cell signalling, sampler kit: phospho-p44/42 MAP Kinase Thr202/Tyr204; 1:5000), GSK3β (Cell signalling #9315; 1:1000), phospho-GSK3β (Cell signalling #9323; 1:1000), S6 (#2212, Cell signalling, Boston, MA; 1:5000), phospho-S6 (Cell signalling #2215; 1:5000). Band intensity was quantified using ImageQuant software (GE Healthcare).

For immunofluorescence (IF) staining of FGFR3, 5μm FFPE sections were subjected to antigen retrieval with citrate buffer for 8 min. Primary antibodies were: FGFR3-N (1:400, sc-13121, Santa Cruz Biotechnology), FGFR3-C (1:2000, sc-123, Santa Cruz Biotechnology), TACC3-N (1:600, ab134153, Abcam), and TACC3-C (1:300, NBP1-01032, Novus Biological). Secondary biotinylated antibodies were used at 1:50,000 followed by streptavidin and TSA Cy3-conjugated. Nuclei were counterstained with DAPI. For immunohistochemical analysis (IHC) of FGFR3 expression, antigen retrieval was performed for 12 min and FGFR-3 antibody (sc-13121, Santa Cruz Biotechnology) was diluted 1:500. Biotinylated anti-mouse antibody (1:30,000) and streptavidin were added before incubation with the chromogen. Nuclei were counterstaining with hematoxylin.