Expression of IIb containing the Gln747 → Pro (Pro747) mutation in transfected cells. (A) Immunoprecipitation analysis of biotin surface-labeled transfected cells. Wild-type or the mutant Pro747 form of IIb cDNA was cotransfected with wild-type β3 cDNA into 293 cells. The transfected cells were surface labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP2 (IIbβ3 complex-specific MoAb). Precipitates were separated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. After transferring to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence. (B) Immunoblot analysis of transfected cells. The transfected cells were lysed and separated by 6% SDS-PAGE under reducing conditions 2 days after transfection. After transferring to a nitrocellulose membrane, IIb and β3 were detected with polyclonal anti-IIbβ3 antisera.
