Two hundred nanograms of recombinant FGFR3 was incubated for 30 min at 30°C with recSHIP2 (SignalChem) as a substrate in 50 μl of kinase buffer [60 mM Hepes (pH 7.5), 3 mM MgCl2, 3 mM MnCl2, 10 μM Na3VO4, and 1.2 mM dithiothreitol] supplemented with 10 μM ATP. For the SHIP2 activity assay, 300 ng of FGFR3 and 400 ng of SHIP2 were incubated with 50 μM PtdIns(3,4,5)P3 (Echelon Biosciences) for 30 min at 30°C. The reaction was then incubated with BIOMOL Green Reagent (Enzo Life Sciences), and optical density at 620 nm was determined by a spectrophotometer. Crystal violet staining was carried out as described (78).

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