Fig. 5.

Western blotting for confirmation of SYK substrates through in vitro kinase assay and in vivo phosphorylation changes in response to BCR stimulation in DG-75 cells. For in vitro kinase assay, individual proteins were immunoprecipitated from cell lysates. The immune complex was then incubated with in a kinase reaction containing purified SYK kinase. For BCR stimulation, DG75 B cells were treated without (IgM-) or with (IgM+) anti-IgM antibodies. Individual proteins were directly isolated from cell lysates. The reaction mixtures were separated by SDS-PAGE and blotted with antigen antibodies and anti-phosphotyrosine antibody (4G10).