Ligation of HLA-DR by Hu1D10 promotes phosphorylation of both Syk and AKT. (A) CLL cells were treated with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for the indicated times. Cells were immediately lysed, immunoprecipated (IP) with anti-Syk/protein A-G agarose beads. Immunoblotting of the IP product was performed with antityrosine antibody (monoclonal antibody 4g10). Gels were stripped and then reprobed with anti-Syk. A representative patient is shown here demonstrating an increase in Syk tyrosine phosphorylation following Hu1D10 treatment. Panel B summarizes the results from 10 different patient samples. The intensity ratios are determined from the ratio of 4g10 band intensity to the Syk band intensity. Values are normalized to the value at time zero. (C) CLL cells were treated with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for the indicated times. Cells were immediately lysed and assessed for changes in AKT activity by determination of Ser473 phosphorylation through immunoblot analysis. A representative patient is shown here demonstrating an increase in phosphorylation of AKT following Hu1D10 treatment. The positive control is Jurkat cell lysate. Panel D summarizes the immunoblot data from 7 different patient samples. (E) Five samples were treated for 0, 5, 15, and 60 minutes with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL). Using the techniques outlined, normalized ratios of Syk-P/Syk and AKT-P/AKT band intensities were determined for each sample at the noted times. For each sample at a given time point the value of the AKT-P and Syk-P ratio is plotted. By definition, the x, y values at time zero equals 1, so these values are not plotted. As indicated by the positive correlation coefficient (R² = 0.53), phospho-AKT levels rise as phospho-Syk levels rise.