Surface expression of α_vβ₃mutants in transiently transfected 293 cells.

(A) The surface expression of transfected α_vβ₃ was analyzed 2 days after transfection by flow cytometry. Cells expressing WT or mutant α_vβ₃ were incubated with 5 μg/mL α_vβ₃ complex-specific mAb LM609 for 30 minutes on ice and then washed once. Bound antibodies were detected by FITC-conjugated goat F(ab′)₂ antimouse IgG. Relative amounts of the binding were normalized to a 100% value for LM609 binding to cells expressing WT α_vβ₃. The results are representative of 3 separate experiments. (B) Immunoprecipitation showing the surface expression of transfected α_vβ₃. Transiently transfected cells were surface-labeled with sulfo-NHS-biotin and lysed in the lysing buffer containing 1% Triton X-100. WT or mutant α_vβ₃ was precipitated with LM609 (specific for the α_vβ₃ complex) and separated on a 6% sodium dodecyl sulfate polyacrylamide gel under reducing conditions. After transfer, a membrane was incubated with peroxidase-conjugated avidin and developed with chemiluminescence. The results are representative of 3 separate experiments.