The D1 mutant of 2B4 binds only SAP. 293T cells were transfected with the indicated 2B4 constructs in combination with SHP-1 (A), SHP-2 (B), Csk (C), or SAP (D). Cells were left untreated or treated with pervanadate (Perv), lysed, and immunoprecipitated (IP) with a control antibody (IgG1) followed by an antibody against 2B4. The immunoprecipitate was then analyzed by Western blotting using the indicated antibodies. Blots were reprobed with an antibody against 2B4 to check for equal immunoprecipitation of the different 2B4 mutants (a representative blot from the experiment in panel B is shown in panel E) followed by antiphosphotyrosine blotting to show 2B4 phosphorylation (a representative blot from the experiment in panel B is shown in panel F). The antiphosphotyrosine blot of panel D showed 2B4 wild-type (wt) phosphorylation also in the absence of pervanadate treatment (data not shown). IgH indicates immunoglobulin heavy chain.