![Fig. 4. Pulse-chase analysis of transfected cells expressing control and mutant αIIbβ3 receptors. / Cells were cotransfected with cDNA constructs expressing control or mutant (Val298Phe and Ile374Thr) αIIb and control β3 subunits for 36 hours. Following preincubation in methionine/cysteine-free medium, cells were labeled with 35S-methionine/cysteine (300 μCi/well [11.1 MBq]) containing medium for 15 minutes and chased in medium containing methionine and cysteine (1 mg/mL each). Cells were harvested at the indicated time points. Using equivalent TCA precipitable counts (about 1.5 × 106 counts/sample), cell lysates were immunoprecipitated using a combination of αIIb-specific mAbs, B1B53031 and M-148 (Santa Cruz Biotechnology; 4 μg each/sample) that recognize pro-αIIb and mature αIIb subunits. Samples were electrophoresed under reduced conditions and dried gels were exposed to film. Bands representing pro-αIIb, mature αIIb, and β3 are shown by arrows.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/101/6/10.1182_blood-2002-07-2266/4/h80633989004.jpeg?Expires=1723742771&Signature=EVAT473Qnnn2DTker5oa6w0UddKUwE5LK1ppnyVY-~uAkwRQP~8ETs4ONtEailItN3RGFxtTqrBLEIRvQdtio5JAW8FXnP-2egbpbSgMV9gkJsMTKaiLDb7lESUMXrHQU2AVzJKNOQkzWrDpoL0eiH~BikIyeh-qeC3Qfk09j-rYnPNxPhyLQe4uSEidntHDkq8qpFgKZ15jRF2bmfFacljtAQJdZV218Uwfcm13Lg36277eRo3sII5aEjZwVc46OEQU0yE5UN06m4GIVzY5NFZkPhmA1uHJsTnoSBkL4QDEqFNhGxTmlwLdtmAwPTwqhhYQE8S6kWYnczPFkdHMTQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Pulse-chase analysis of transfected cells expressing control and mutant αIIbβ3 receptors.
Cells were cotransfected with cDNA constructs expressing control or mutant (Val298Phe and Ile374Thr) αIIb and control β3 subunits for 36 hours. Following preincubation in methionine/cysteine-free medium, cells were labeled with 35S-methionine/cysteine (300 μCi/well [11.1 MBq]) containing medium for 15 minutes and chased in medium containing methionine and cysteine (1 mg/mL each). Cells were harvested at the indicated time points. Using equivalent TCA precipitable counts (about 1.5 × 106 counts/sample), cell lysates were immunoprecipitated using a combination of αIIb-specific mAbs, B1B530,31 and M-148 (Santa Cruz Biotechnology; 4 μg each/sample) that recognize pro-αIIb and mature αIIb subunits. Samples were electrophoresed under reduced conditions and dried gels were exposed to film. Bands representing pro-αIIb, mature αIIb, and β3 are shown by arrows.
