Fig. 3.

(A) Confirmation of Syk kinase substrates through in vitro kinase assay and endogenous phosphorylation changes in response to IgM stimulation in DG-75 cells. For in vitro kinase assay, individual proteins were isolated by immunoaffinity purification from cell lysate and kinase reaction was carried out directly on the beads with ATP and Syk. The reaction mixture was separated by SDS-PAGE and analyzed by Western blotting (WB) with the indicated antibodies and antiphosphotyrosine antibody (4G10). For IgM stimulation, individual proteins were isolated from cell lysate, directly separated by SDS-PAGE, and analyzed by WB with the indicated antibodies and antiphosphotyrosine antibody (4G10); (B) in vitro kinase assays of GST-Nek9 full length, truncated, and mutant (Y520I/Y521D) with or without the presence of Syk.