Measurement of intracellular signaling. (A) Western blot analysis of protein tyrosine phosphorylation was performed using fresh BCL₁ cells as described in “Materials and methods.” Lane 1 indicates anti-μ (IgM heavy chain); 2, anti-Id; 3, anti-MHCII; 4, anti-CD19; 5, anti-CD22; 6, anti-CD38; 7, control; and 8, molecular weight marker. Anti-μ and anti-Id as single mAb demonstrate intracellular signaling through the surface Ig receptor (BCR) as measured by the induction of cellular protein tyrosine phosphorylation. Hyper–cross-linking of primary mAb with sheep antirat IgG demonstrates signal transduction in anti-CD19 and anti-CD22 as well as via the BCR. (B) Addition of radiation enhances the level of signaling observed through these molecules. (C) Immunoprecipitation of phosphorylated proteins using 4G10, followed by immunoblotting with anti-Syk mAb, revealed that the approximately 70-kDa phosphorylated protein is likely to be Syk. (D) Measurement of intracellular calcium flux in lymphoma cells after hyper–cross-linking mAb. πBCL₁ lymphoma cells were loaded with Indo-1-AM and then incubated with either mAb alone (2 μg/mL) or mAb plus an appropriate secondary polyclonal anti-IgG (20 μg/mL). Profiles of FL5/FL4 are shown versus time. Control or irrelevant IgG was a nonbinding anti-Id mAb (to the A31 lymphoma) in both sets of experiments.