Generation of β3 knockin mice. (A) Wild-type β3^+/+ (WT) and mutant β3 cytoplasmic domain sequences. β3(Δ760-762) lacks the 3 C-terminal residues of β3 (arginine-glycine-threonine [RGT]), while in β3/β1(glutamic acid–glycine-lysine [EGK]) those residues have been replaced with the respective 3 C-terminal residues of β1. (B) Gene-targeting strategy. A 7.2-kb targeting vector for the mouse β3 gene contained a Neo cassette flanked by 2 lox P sequences between β3 exons 14 and 15. Either of the β3 mutations was introduced into exon 15. B, BamHI; E, EcoRI; X, XhoI. (C) Southern blot analysis with a 3′ probe of R1 embryonic stem (ES) cell genomic DNA transfected with the β3(Δ760-762) targeting vector and digested with EcoRI. (D) PCR genotyping of final floxed β3 cytoplasmic domain mutants. (E) Mouse platelet lysates blotted with antibodies recognizing the extracellular domain of β3 (anti-β3) or the β3 C-terminus (anti-β3 C-terminus). (F) Platelet lysate (10 μg) from β3^+/+, β3^+/−, and β3(Δ760-762) mice were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotted with antibody to β3 or c-Src. (G) Surface expression of αIIbβ3 was determined by flow cytometry using an antibody to αIIb. Data are expressed as mean fluorescence intensity in arbitrary units plus or minus standard error of the mean [SEM] (n = 5-13).