Characteristics of primaquine-induced endovesicles. (A) Micrograph of a live mature erythrocyte containing LY+ primaquine-induced endovesicles. Single merged optical section of differential interference contrast (DIC) and green fluorescent (LY) channels; 60×/1.42 oil objective (Olympus). Scale bar equals 3 μm. (B) Association of LY with membrane fractions 1 through 5 from primaquine-treated erythrocytes separated by sucrose density centrifugation and quantified by immunoblotting with anti-LY antibodies and densitometry. Fraction numbers are indicated (1, top; 5, bottom). Bars show total LY signal in each fraction divided by total LY signal in the starting material (T) expressed as a percentage. Error bars indicates standard deviation of duplicate measurements. (C) Cholesterol-protein ratios of endovesicle fractions 1 through 5. Cholesterol and protein levels were measured and ratios calculated as described for erythrocyte DRMs4 ; hemoglobin-free erythrocyte membranes were used as reference values. T indicates starting material; WM, hemoglobin-free white membranes. (D) Immunblotting of erythrocyte endovesicle membrane fractions using specific antisera for the indicated human proteins (flotillin-2, stomatin, actin, spectrin) and specific secondary antibodies. T indicates total lysate. Fractions 2 and 3 were enriched in the raft protein flotillin-2, mildly enriched for human stomatin, and contained lesser amounts of cytoskeletal proteins actin and spectrin.
