Association of signaling molecules with the cytoplasmic tail of 2B4. (A) A fusion protein of the 2B4 cytoplasmic tail with GST was produced either nonphosphorylated (GST-2B4) or phosphorylated (GST-2B4-P) and coupled to beads. GST alone was used as a control. Beads were incubated with the lysate of NK-enriched PBLs and associated proteins were identified by Western blotting using the indicated antibodies. (Bi) NK cells were mixed with the CD48⁺ target cell 721.221 for 0 or 5 minutes. 2B4 was immunoprecipitated and analyzed by Western blotting with an anti-SAP monoclonal antibody (top). The blot was reprobed with an antibody against 2B4 (middle) and against phosphotyrosine (bottom) to demonstrate 2B4 phosphorylation and equal immunoprecipitation. (ii) Equal SAP expression was shown by anti-SAP blotting of the lysate.