Immunoprecipitation analysis of −889PlA2β3 transduced CD34+cells from patient RS▵9β3 and EAY115Cβ3.
Cells (5 × 105) were collected 10 and 15 days, respectively, after transduction and detergent lysates were immunoprecipitated with 10 μg of an αIIbβ3 complex-specific antibody (AP2). The complexed proteins were separated on a 7% SDS-PAGE gel under nonreducing conditions. Immunoanalysis using polyclonal antibodies to αIIb and β3 followed by chemiluminescence detection showed that transduction with the −889PlA2β3 vector resulted in the synthesis of detectable levels of β3and αIIb (arrows on right), whereas untransduced samples did not have detectable protein. Molecular mass markers are in kilodaltons (left). Additional bands appearing equally in each lane are nonspecific background resulting from chemiluminescence detection using a murine monoclonal antibody for immunoprecipitation and rabbit polyclonal antibodies and a horseradish peroxidase-conjugated donkey antirabbit antibody for analysis.
