Murine anti–β3 integrin antibodies were generated after immunizing β₃^-/- female mice with wild-type mouse platelets. (A) WT mouse platelets were incubated with a 1:100 dilution of sera from β₃^-/- mice immunized either 2 or 4 times weekly (dotted line indicates 2 times; bold line, 4 times) or preimmune sera (filled area) and stained with FITC-conjugated goat anti–mouse IgG or rat anti–mouse IgG₁ or IgG_2a. β₃^-/- platelets or WT red blood cells (RBCs) were used as negative controls. (B) Titration of IgG antibody in preimmune sera of β₃^-/- mice (□), and sera of mice after 2 (○) or 4 (♦) WT platelet transfusions. (C) Western blot of platelet lysates with both control anti–β₃ integrin antibody sc-6627 and antisera of mice after 4 WT platelet transfusions. No β₃ integrin band was recognized by sera from nonimmunized β₃^-/- mice in the negative control. (D) β₃ integrin was immunoprecipitated from platelet lysates using either antisera from this study or the monoclonal antibody JON2 (anti-mouse β₃ integrin) or p0p3 (anti-mouse GPIbα, negative control). The β₃ integrin immunoreactive band was detected by both the antisera (left panel) and the positive control antibody sc-6627 (right panel) in immunoblot analysis. (E) Thrombocytopenia was induced in WT BALB/c mice by 100 μL antisera and their dilutions from the β₃^-/- mice transfused 2 times (right panel) or 4 times (left panel) with WT platelets. PBS was used as a control. n = 3 in each group. Data are represented as means ± SEM.