Western blot analysis of globin deposition on the membranes of in vivo–aged biotinylated dog RBCs. Positive (biotinylated) RBCs were isolated from dog no. 5005 at the indicated times post-biotinylation by magnetic cell sorting. (A) Ghost membranes were prepared as described in the Materials and Methods. At the end of the study, membranes (20 μg) were electrophoresed, transferred to nitrocellulose, and analyzed for globin by immunoblotting as described in Materials and Methods. The lane marked Hb contains 5 μg of soluble Hb obtained from a dog RBC lysate. (B) Results of scanning densitometry of the dimeric globin band seen in the immunoblot in (A). The integrated area of the globin dimer band is plotted as a function of the minimum age of the biotinylated cells. A similar increase in membrane-bound globin appeared on the membranes of cells isolated from dog no. 1630 (data not shown).