Effect of the Pro747 mutant on _IIbβ₃ biosynthesis. (A) Association of wild-type or the Pro747 mutant pro_IIb with wild-type β₃. Wild-type or the Pro747 mutant _IIbβ₃ transfected cells were labeled with 0.2 mCi/mL of [³⁵S]-methionine for 120 minutes and total cellular lysates were prepared. Subunit association was assessed by immunoprecipitation with TP80 (_IIb-specific MoAb) or AP3 (β₃-specific MoAb). Precipitates were separated by 6% SDS-PAGE under reducing conditions. (B) Pulse chase analysis of the stability of wild-type and Pro747 mutant pro_IIb subunit. Wild-type or Pro747_IIb cDNA was transfected into 293 cells without the wild-type β₃ cDNA, and cells were labeled with 0.4 mCi/mL of [³⁵S]-methionine for 30 minutes and chased with media containing 50 μg/mL of nonradioactive methionine for various periods of time, as indicated. Immunoprecipitation was performed using TP80. Precipitates were separated by 6% SDS-PAGE under reducing conditions. (C) Pulse chase analysis of wild-type or the Pro747 mutant _IIbβ₃ in transfected cells. Wild-type or Pro747 _IIbβ₃ transfected cells were labeled with 0.4 mCi/mL of [³⁵S]-methionine for 30 minutes and chased with media containing 50 μg/mL of nonradioactive methionine for various periods of time, as indicated. Immunoprecipitation was performed using TP80. Precipitates were separated by 6% SDS-PAGE under reducing conditions. This figure shows a representative of six separate experiments. (D) Densitometric analysis of the kinetics of biosynthesis of _IIbβ₃ shown in (C). The bands corresponding to pro_IIb (□), _IIb (◊), and β₃ (○) were analyzed by scanning densitometry. The results were normalized relative to dye-front band at each lane.