Fyn is recruited to the phosphorylated 2B4 receptor and Csk and Fyn can phosphorylate 2B4. (A) A blot shown in Figure 3C was stripped and reprobed with a rabbit polyclonal anti-Fyn antibody. (B) GST fusion proteins were incubated with the lysate from NK-cell-enriched PBLs as described in Figure 4 and analyzed by Western blotting using a monoclonal anti-Fyn antibody. (C) 293T cells were transfected with 2B4 alone or in combination with Fyn or Csk. Cells were lysed and immunoprecipitated (IP) with a control antibody (IgG1) followed by an antibody against 2B4. The immunoprecipitate was then analyzed by antiphosphotyrosine Western blotting (top blot) and reprobed with an antibody against 2B4 to show equal immunoprecipitation (bottom blot). (D) 293T cells were transfected with the indicated 2B4 mutants alone (-) or in combination with Fyn (+). Y5F is a control mutant affecting a tyrosine at the end of the cytoplasmic tail of 2B4, which is not part of an ITSM sequence. In the Y1234F mutant all ITSMs are mutated. Cells were lysed and immunoprecipitated (IP) with a control antibody (IgG1) followed by an antibody against 2B4. The immunoprecipitate was then analyzed by antiphosphotyrosine Western blotting (top blot) and reprobed with an antibody against 2B4 to show equal immunoprecipitation (middle blot). Similar expression of Fyn was confirmed by anti-Fyn Western blotting of the different lysates (bottom blot).