Analysis of mutant protein expression. A. Surface expression of αIIbβ3 on HEK293 cells expressing normal or mutant αIIbβ3 receptors as judged by the binding of mAb 10E5.

<p>1 million cells in 100 µl were incubated with 5 µg/ml Alexa488-conjugated mAb 10E5 (black line) or as a control for non-specific binding, 5 µg/ml Alexa488-conjugated mAb 10E5 in the presence of excess (125 µg/ml) unlabelled 10E5 (gray line; background). <b>B. Disulfide-bonded αIIbβ3 heterodimer formation on the cell surface of XS-O mutants.</b> HEK293 cells expressing normal αIIbβ3 or XS-O mutants were biotinylated and lysed, and lysates were immunoprecipitated with anti-αIIbβ3 complex mAb 10E5. After SDS-PAGE and protein transfer, biotinylated proteins were identified with streptavidin-HRP. <b>Left panel.</b> Non-reduced SDS-PAGE analysis of normal αIIbβ3 and XS-O mutants. <b>Right panel.</b> SDS-PAGE analysis of normal αIIbβ3 and XS-O mutants under reducing conditions (10% β-mercaptoethanol). <b>C and D. Mass spectroscopy identifies unique, predicted peptides from XS-O mutants. </b><b>C.</b> Purified protein from mutant 321/358 was digested with trypsin and analyzed by LC-MS/MS. A peak at m/z = 488.2569 corresponded to the predicted disulfide linked MH₄³⁺ ion of peptide VELC(-VR)-CLAEVGR. <b>D.</b> Purified protein from mutant 321/360 was digested with trypsin and ASP-N, followed by LC-MS/MS analysis. A peak at m/z = 653.2639 corresponded to the predicted disulfide linked MH₄⁺ ion of EVC-CLA.