FIGURE 6.
Quantification of free cysteines in αIIbβ3 mutants. A, cells co-transfected with αIIb and the indicated β3 mutants were labeled with biotin-BMCC, lysed, and subjected to immunoprecipitation with AP3 antibody, non-reducing SDS-PAGE, and Western blotting with HRP-conjugated streptavidin to detect biotin-BMCC-labeled free cysteines or anti-His Tag antibody and HRP-conjugated rabbit anti-IgG to detect the His6-tagged β3-subunit. The β3V332C mutant and wild-type transfectants pretreated with 10 mm DTT for 1 h at 37 °C served as positive controls. B, number of free cysteines was calculated from the intensity ratio of the biotin-BMCC and anti-His bands after subtraction of the intensity of a band of same area.