R406 inhibits BCR signaling in CLL cells. (A) Displayed are immunoblots from CLL cells from 2 representative patients who were either unstimulated (control), or stimulated for 10 or 20 minutes with anti-IgM in the presence or absence of R406. These different conditions are displayed above the immunoblots. At the indicated time points, the cells were lysed and probed with mAbs to the antigens that are displayed on the left side. “P” indicates immunoblotting for the active, phosphorylated form, whereas “T” represents the nonphosphorylated, total amount of the respective proteins. We found that constitutive and anti-IgM–induced Syk activation, p44/42 mitogen-activated protein kinase (ERK), and AKT activation were inhibited by R406. Relative protein levels were determined by densitometry, normalized to the corresponding control, and are displayed below each of the phosphoprotein bands. (B) Calcium mobilization is another signaling response typically induced in B cells after BCR triggering. In this figure, CLL cells were labeled with the fluorescent calcium-indicator Fluo3-AM, and preincubated with or without R406. Then the CLL cells were stimulated with anti-IgM, and the fluorescence intensity was recorded over time. The lines represent the changes in fluorescence intensity (on the vertical axis) over time (horizontal axis) for control CLL cells (black lines) or cells preincubated with R406 (gray lines). Displayed is the anti-IgM–induced calcium mobilization in 4 different patients (labeled CLL#1-4), with a detectable calcium mobilization in the controls, and an abrogated calcium mobilization in R406-pretreated CLL cells.