3BP2 is phosphorylated by BCR engagement. (A) Lysates of various B-cell lines were analyzed by Western blot for 3BP2 expression. ERK2 expression was used as a control to quantify protein levels in each lane. (B) Daudi cells were transfected with a plasmid encoding HA-tagged 3BP2 or empty vector. HA-immunoprecipitates were then probed with anti-3BP2 or anti-HA antibodies. The membrane was reprobed with anti-HA antibody to control 3BP2 protein expression. (C) BCR cross-linking results in 3BP2 tyrosine phosphorylation. Daudi cells were stimulated for 5 minutes with anti-IgM antibody (2 μg/mL). Immunoprecipitates were prepared with anti-3BP2 antibody and probed with antibodies to phosphotyrosine and 3BP2. (D) COS-1 cells were transiently cotransfected with expression plasmids encoding HA-tagged 3BP2 and Fyn, Syk, or Btk constructs. HA-immunoprecipitates were probed with antiphosphotyrosine antibody. The membrane was reprobed with anti-HA antibody to control 3BP2 protein expression.