Figure 4.

ER-27319 inhibits Syk activity in activated intact cells but does not inhibit activated Syk in vitro. IgE-sensitized RBL-2H3 cells were incubated in the presence or absence of 30 μM ER-27319 (ER) or 100 nM piceatannol (Pic) before stimulation for 3 min with antigen (Agn). Cells were lysed and Syk was immunoprecipitated. Immune complexes were incubated with GST-HS1 in an in vitro kinase assay (lanes 1–4). In lanes 5–7, ER-27319 or piceatannol was added to the assay mixture containing Syk immune complexes from activated cells. Proteins were resolved by SDS/PAGE. Western blot analyses with anti-phosphotyrosine, anti-Syk, and anti-GST were performed sequentially. One of three representative experiments is shown.