SAP blocks the binding of SHP-1, SHP-2, and Csk to 2B4. 293T cells were transfected with 2B4, SAP, and SHP-1 (A), SHP-2 (B), or Csk (C) as indicated and 2B4 phosphorylation was induced by pervanadate treatment. Samples were immunoprecipitated with a control antibody (IgG1) or with an anti-2B4 antibody and analyzed by Western blotting using the indicated antibodies (top panels). Blots were reprobed with an antibody against 2B4 to check for equal immunoprecipitation of 2B4 followed by antiphosphotyrosine blotting to show 2B4 phosphorylation (a representative blot is shown in panel D). The lysate of the different transfections was analyzed by anti-SAP, SHP-1, SHP-2, or Csk Western blotting to ensure equal expression (bottom panels).