![Fig. 7. Effect of the Pro747 mutant on IIbβ3 biosynthesis. (A) Association of wild-type or the Pro747 mutant proIIb 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 proIIb subunit. Wild-type or Pro747IIb 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 proIIb (□), IIb (◊), and β3 (○) were analyzed by scanning densitometry. The results were normalized relative to dye-front band at each lane.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/92/8/10.1182_blood.v92.8.2750/5/blod42033007w.jpeg?Expires=1726825469&Signature=knfP3S-bhxiRmCmQH~nVvHNON9UWBZS40aBK3LJaahOOCdY7dY~Vtj6XGJCss1P6vpIz1HKIoZ1ewoj-gUrDX1L0kjwvhBBcC5bVBDX5RXuamptwL-43CYEmoyXDZN3l3IzWsxDXrZ5SiWo3IhSIIr7khZRSIGy-v3REvYI1mVBzEvtiaXzj3wH9s6rmN~lzU94-iXWPyDP2uuRdHxuRjsZQ~ZrjJ4rBvheH~q~K5cU7XNRxA4bkzoto4YMqgxe7FRp432xnu3VUZozQskwF7DDi2onFcZP0abbGCVy9TeulV9E5g7~-3qncVuFG6UZEk0a1UaSHfWiGLoIFRYzyNA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of the Pro747 mutant on IIbβ3 biosynthesis. (A) Association of wild-type or the Pro747 mutant proIIb 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 proIIb subunit. Wild-type or Pro747IIb 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 proIIb (□), IIb (◊), and β3 (○) were analyzed by scanning densitometry. The results were normalized relative to dye-front band at each lane.
