Effects of His280Pro β₃ missense mutation on the expression of α_IIbβ₃ and α_vβ₃ in 293 cells.

(A) Flow cytometric analysis of α_IIbβ₃ on the transfected cell surface. Wild-type or His280Proβ₃cDNA was cotransfected into 293 cells with wild-type αIIb cDNA and GFP expression vector pEGFP-C1. The binding of AP2, TP80, and PAC-1 with PT25-2 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results are representative of at least 3 separate experiments. (B) Immunoprecipitation analysis of biotin surface-labeled transfected cells. The transfected cells were surface-labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP3. Precipitates were separated by 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. After transfer to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence. (C) Immunoblot analysis of transfected cells. The transfected cells were lysed and separated by 6% SDS-PAGE under reducing conditions 2 days after transfection. After transfer to a nitrocellulose membrane, αIIb and β₃ were detected with a 1:10 000 dilution of rabbit polyclonal anti-α_IIbβ₃ antibodies. (D) Flow cytometric analysis of α_vβ₃ on the transfected cell surface. Wild-type or His280Proβ₃ cDNA was transfected into 293 cells (i) in the absence or (ii) in the presence of wild-type αv cDNA. The binding of LM609 (specific for α_vβ₃ complex) or LM142 (specific for αv) to the transfected cells was analyzed by flow cytometry 2 days after transfection. MOPC21 was used as a negative control (dotted line).