Activating FGFR2 breast cancer mutations.

<p><b>A)</b> Representative Western blot showing increased tyrosine kinase activities of FGFR2-IIIb breast cancer mutants compared to tyrosine kinase activities of wt FGFR2-IIIb (WT), of a kinase defective (KD) FGFR2-IIIb mutant, and of a Pfeiffer syndrome gain-of-function mutant (K642R, previously named K641R according to another reference sequence) as controls. HEK293 cells were transiently transfected using lipofectamine with pRK5 vectors containing cDNA coding for wt FGFR2-IIIb or FGFR2-IIIb variants containing the indicated amino acid substitution. Untransfected cells served as negative control (−). Lysates from cells were subjected to immunoprecipitation with anti-FGFR2 antibodies (Bek(C-17)) followed by SDS-PAGE and immunoblotting (IB) with anti-FGFR2 or anti-p-Tyr antibodies (PY99). <b>B</b>) Representative Western blots showing increased substrate phosphorylation by FGFR2-IIIb breast cancer mutants compared to substrate phosphorylation by wt FGFR2-IIIb (WT), a kinase defective (KD) FGFR2-IIIb mutant, and a Pfeiffer syndrome gain-of-function mutant (K642R, previously named K641R according to another reference sequence) as controls. For transient transfection calcium phosphate was used. Untransfected cells served as negative control (−). HEK293 cells expressing FGFR2-IIIb as well as untransfected cells were stimulated with FGF1. Lysates of unstimulated or FGF1-stimulated cells were subjected to SDS-PAGE followed by immunoblotting with anti-p-FRS2, anti-p-MEK1/2, anti-p-STAT3 and anti-ß-Actin as loading control.