FIGURE 4. Kinetics of wild-type Cas phosphorylation at different substrate concentrations.

A, WT Cas was incubated in the presence of 50 nm v-Src at 30 °C in kinase assay buffer. Reactions were started by the addition of 100 µm unlabeled ATP plus [γ-³²P]ATP (5 µCi). Aliquots were withdrawn at the indicated times and analyzed by 8% SDS-PAGE and autoradiography. B, incorporation of ³²P into Cas was measured by exposing the dried gels to a PhosphorImager screen (GE Healthcare) and quantifying the band intensity using ImageQuant software. A graph of velocity (PhosphorImager units/min) versus substrate concentration is shown. The data were fit to the Michaelis-Menten equation using nonlinear regression analysis.