Analysis of proximal and distal signaling events in CLL cells derived from CLL patients. (A) CLL cells were left untreated (black histogram) or treated with anti-IgM (gray histogram) for 15 minutes. They were then fixed and stained with an FITC-conjugated antiphosphotyrosine antibody (PY20), and fluorescence was measured by flow cytometry. (B) CLL cells were left untreated or treated with anti-IgM as indicated for 1 hour. Nuclear extracts were prepared and NF-κB activity was evaluated by electrophoretic mobility shift assay; only the shifted bands are shown. (C) The percentage change in protein tyrosine phosphorylation and NF-κB activity (before and after ligation with anti-IgM) was calculated. The percentage change in NF-κB correlated with percentage change in protein tyrosine phosphorylation (r² = 0.2, P = .03). (D) Cell apoptosis was measured after 24 hours using annexin V binding and PI exclusion in untreated CLL cells and cells treated with anti-IgM. The percentages shown in the upper right quadrant of each dot plot denote the total percentage of apoptotic cells in the upper and lower right quadrants. (E) The relationship between the percentage change in NF-κB and the percentage change in apoptosis was investigated, and a significant inverse correlation was detected (r² = 0.67, P < .001). (F) ZAP-70 expression was associated with NF-κB activation after exposure to anti-IgM (P = .003).