Identification of proteins in erythrocyte microvesicles and nanovesicles.

(A) Erythrocytes were treated with Ca⁺⁺/A23187, and microvesicles and nanovesicles were prepared according to method A. Aliquots of the erythrocyte membranes (lane 1), microvesicles (lane 3), and nanovesicles (lane 3), normalized to AChE activity, were analyzed by 12% polyacrylamide gel electrophoresis/silver staining (upper panel) and immunoblotting (lower panel) as indicated. (B) Vesicles were prepared according to method B. Aliquots, normalized to AChE activity, of the erythrocyte membranes (lane 1) and vesicles obtained after low-speed centrifugation for 10 minutes (lane 2) and 20 minutes (lane 3) and ultracentrifugation (lane 4) were analyzed as in panel A. CA indicates carbonic anhydrase; Hb, hemoglobin.