ZAP-70 is not phosphorylated on positive regulatory tyrosine residues following BCR stimulation in CLL B cells. (A) Immunoblotting analysis of total cellular extracts from 3 ZAP-70⁺ (G25, G56, and G10) and 2 ZAP-70⁻ CLL samples (G17 and G98) using the phospho-ZAP-70^Tyr319/Syk^Tyr352 antibody. Cells were stimulated with anti-IgM, as indicated. Jurkat T cells stimulated for 5 minutes with anti-CD3 antibody were used as a positive control for ZAP-70 activation. BJAB B cells stimulated for 5 minutes with anti-IgM were used as a positive control for Syk activation. The same membrane was reprobed with anti–ZAP-70, anti-Syk, and antitubulin antibodies, as indicated in the bottom panels. Arrows indicate the expected position of the ZAP-70, Syk, and SykB proteins. (B) Immunoblotting analysis with phospho-ZAP-70^Tyr319/Syk^Tyr352 antibody of ZAP-70 and Syk proteins immunoprecipitated from unstimulated or anti-IgM–stimulated ZAP-70⁺ CLL B cells (sample G4) is shown in the top panel. Jurkat T cells unstimulated or stimulated with anti-CD3 were used as a positive control for ZAP-70 phosphorylation, whereas unstimulated or anti-IgM–stimulated BJAB B cells were used as a positive control for Syk phosphorylation. The total amount of immunoprecipitated ZAP-70 and Syk protein is shown in the bottom panels. (C) Immunoblotting analysis of total cellular extracts from ZAP-70⁺ and ZAP-70⁻ CLL B cells, BJAB B cells, and Jurkat T cells using the phospho-ZAP-70^Tyr493 antibody, which also cross-reacts with phospho-Syk^Tyr526. Stimulation with anti-IgM or anti-CD3 was performed for the indicated times. (D) Immunoblotting analysis of immunoprecipitated ZAP-70 and Syk from unstimulated or anti-IgM–stimulated ZAP-70⁺ CLL B cells. Jurkat T cells stimulated with anti-CD3 are shown as a positive control for ZAP-70 activation. Membranes were probed with the same phospho-ZAP-70/Syk antibody as in panel C (top panels) and reprobed with anti–ZAP-70 (middle panels) and anti-Syk (bottom panel).