Figure 2.

Properties of purified SYK isoforms. A, Western blotting illustrating the phosphorylation levels of the four SYK isoforms (lanes 1 and 5, 50 ng of GST-upSYK; lanes 2 and 6, 50 ng of GST-pSYK; lanes 3 and 7, 36 ng upSYK, and lanes 4 and 8, 36 ng of pSYK). Lanes 1–4 were probed with mouse anti-human SYK to ensure equal loading of phosphorylated (designated as p) versus unphosphorylated (designated as up) SYK. The corresponding lanes 5–8 were probed with a mouse anti-phosphotyrosine antibody to show phosphorylation levels for each of the four isoforms. The location of a molecular mass standard band migrating at 97 kDa is denoted with an arrow. B, phosphorylation of a generic peptide (Omnia® S/T peptide 7) by each SYK isoform was evaluated in a continuous kinetic Omnia kinase assay. Enzyme, ATP, and peptide concentrations used for all four kinase reactions were 4 nm, 35 μm, and 10 μm, respectively. The phosphorylated product was quantified relative to a standard curve. GST-pSYK (blue circles), pSYK (red triangles), GST-upSYK (green squares), and upSYK (pink crosses) are shown.