Tyr292 phosphorylation and interaction of ZAP-70 with PI3K p85α, c-Cbl, Cbl-b, and Shc. (A) Immunoblotting analysis with phospho-ZAP-70Tyr292 antibody of total cellular extracts from parental BJAB B cells, 2 BJAB clones expressing different levels of ZAP-70 (clone A and clone G), Jurkat T cells, and ZAP-70+ (G25, G56, and G10) or ZAP-70− (G17 and G98) CLL B cells. Cells were stimulated with anti-IgM or anti-CD3, as applicable. (B) Immunoblotting analysis with anti–ZAP-70 antibody of protein complexes immunoprecipitated with PI3K p85α, c-Cbl, Cbl-b, and Shc antibodies. Immunoprecipitations were performed before and after antigen receptor stimulation on parental and ZAP-70–transfected BJAB B cells, Jurkat T cells, and CLL B cells, as indicated. Membranes were reprobed with antibodies against p85α, c-Cbl, Cbl-b, and Shc to evaluate the quantity of immunoprecipitated proteins.
