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Fig. 1. FGFR3 interacts with STAT1 and STAT3. FLAG-tagged wt, FGFR3-K650E or FGFR3-K508M were expressed in HeLa cells (A) or RCS chondrocytes (B) and the whole cell lysates were subjected to WB for indicated molecules (left panel). Actin serves as a loading control. The same lysates were subjected to STAT1 (middle panel) and STAT3 (right panel) immunoprecipitation (IP) followed by WB. Please note that the transfected FGFR3-FLAG was detected by FLAG antibody in A and by FGFR3 antibody in B. (C) FLAG-tagged wt or FGFR3-K650E, immunoprecipitated from CHO cells, or recombinant tyrosine kinase (TK) domain of FGFR3 were subjected to an in vitro kinase assay with recombinant STAT1 as a substrate. The level of STAT1 phosphorylation was determined by WB with antibody recognizing STAT1 only when phosphorylated at Y701. Samples including the FGFR inhibitor SU5402 (20 µM) or those with ATP omitted serve as negative controls for the kinase reaction. The levels of total STAT1 and FGFR3 serve as controls for the substrate and kinase quantity, respectively. Note that the FGFR3 antibody was raised against the extracellular domain of FGFR3 and thus cannot recognize FGFR3-TK. (D) The kinase assay was carried out as described above, using FGFR3-TK as a kinase, and recombinant STAT1 and/or recombinant FRS2 as substrates. The level of STAT1 phosphorylation was determined as described above, whereas FRS2 tyrosine phosphorylation was determined by WB with the 4G10 phosphotyrosine antibody.
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