Analysis of Syk activity and BCR signaling in TCL1 leukemias in vitro. (A) The presence of active Syk protein was investigated by immunoblotting analysis with phospho-specific antibodies on cellular extracts prepared from spleens of mice with TCL1 leukemias. Normal B cells that were purified by negative selection from spleens of wild-type mice were used as controls. (B) Freshly isolated TCL1 leukemia cells were cultured for 90 minutes in the presence of the indicated concentrations of R406. The effect on the basal activity of signaling molecules downstream of Syk was investigated by immunoblotting analysis using phospho-specific antibodies. Syk and ERK served as loading controls. (C) TCL1 leukemia cells were preincubated for 90 minutes with the indicated concentrations of R406 prior to stimulation with anti-IgM antibody for 3 minutes. Cells were collected and analyzed as above.