Analysis of Syk phosphorylation following anti-IgM treatment.

(A) CLL samples were incubated with anti-IgM or control antibody for 2 minutes. Tyrosine-phosphorylated Syk was analyzed by immunoprecipitation (IP) with phosphotyrosine-specific antibody followed by blotting with the Syk-specific antibody (top panel). Total Syk expression was analyzed by direct immunoblotting (bottom panel). Representative results are shown. Table 2 provides summary of data for all samples. (B) Daudi cells or CLL samples were incubated with anti-IgM or control antibody for 2 minutes. Tyrosine phosphorylation on Tyr525/526 of Syk was detected by immunoprecipitation using the Syk phospho-Tyr525/526–specific antibody followed by direct immunoblotting for Syk. Representative results are shown for analysis of 6 CLL samples. Patient 9 is an unmutated sample that showed global tyrosine phosphorylation following anti-IgM treatment, whereas patient 24 is a mutated sample that did not show phosphorylation with anti-IgM.