Figure 1. Two distinct RBC phenotypes of... | American Society of Hematology

Figure 1.

$Two distinct RBC phenotypes of band 3 deficiency in cattle. (A) Scanning electron micrographs and (B) phase-contrast micrographs of RBCs from a healthy control cattle (a) and a type 1 (b) and a type 2 (c) band 3–deficient cattle. For scanning electron micrography found in panel A, whole blood cells were fixed with 1.0% glutaraldehyde in 0.1 M sodium phosphate (pH 7.4) within 1 hour after sampling. RBCs were suspended in PBS and allowed to stand at ambient temperature. After 6 hours, RBCs were examined under phase-contrast microscopy at ×1000 magnification in panel B and the numbers of RBCs possessing prominent protrusions were counted. (C) The top panel indicates a magnification of the boxed area in (b) in panel B. Data are illustrated in percentage of RBCs with protrusions in 200 RBCs and expressed as the means ± SD (n = 4). ∗∗∗∗P < .0001 by 1-way analysis of variance (ANOVA) with the Tukey multiple comparison test. Scale bars, 6 μm (A), 20 μm (B), and 10 μm (C), respectively. (D) Ghost membranes (Ghost) were prepared from 1 × 108 RBCs obtained from a healthy normal animal (Control) and a type 1 (B3-null 1) and a type 2 (B3-null 2) band 3–deficient cattle. Membrane proteins were solubilized in 2% Triton X-100 and the insoluble fractions (Triton shell) were obtained by ultracentrifugation. Proteins in the ghosts and Triton shells were separated on 8% SDS gels followed by staining with Coomassie brilliant blue. The bands indicated by an open arrowhead contain serum albumin in invagination-derived vesicles.14 (E) Filtration-induced hemolysis generated fragmented vesicles in the filtrates. RBCs from the animals were pressurized on the membrane filter and the vesicles in the filtrate were collected by ultracentrifugation and washed once. Proteins in the vesicles were directly solubilized in the sample buffer for SDS-PAGE, and half the volume of the sample was loaded on 8% SDS gels followed by staining with Coomassie blue. Note that the vesicles from type 1 RBCs were dissolved in 5× the volume of buffer and one-tenth of the solubilized sample, that is equivalent to the volume of other samples, was applied to the well. α- and β-spectrin are apparent only in the vesicles from type 1 band 3–deficient RBCs (arrowheads). Migrating positions of α- and β-spectrin, band 3, 4.1R, 4.2, actin, and globins in ghost membranes, including those of size marker proteins found in kDa, are indicated in panels D-E. ns, not significant.$

Two distinct RBC phenotypes of band 3 deficiency in cattle. (A) Scanning electron micrographs and (B) phase-contrast micrographs of RBCs from a healthy control cattle (a) and a type 1 (b) and a type 2 (c) band 3–deficient cattle. For scanning electron micrography found in panel A, whole blood cells were fixed with 1.0% glutaraldehyde in 0.1 M sodium phosphate (pH 7.4) within 1 hour after sampling. RBCs were suspended in PBS and allowed to stand at ambient temperature. After 6 hours, RBCs were examined under phase-contrast microscopy at ×1000 magnification in panel B and the numbers of RBCs possessing prominent protrusions were counted. (C) The top panel indicates a magnification of the boxed area in (b) in panel B. Data are illustrated in percentage of RBCs with protrusions in 200 RBCs and expressed as the means ± SD (n = 4). ∗∗∗∗P < .0001 by 1-way analysis of variance (ANOVA) with the Tukey multiple comparison test. Scale bars, 6 μm (A), 20 μm (B), and 10 μm (C), respectively. (D) Ghost membranes (Ghost) were prepared from 1 × 10⁸ RBCs obtained from a healthy normal animal (Control) and a type 1 (B3-null 1) and a type 2 (B3-null 2) band 3–deficient cattle. Membrane proteins were solubilized in 2% Triton X-100 and the insoluble fractions (Triton shell) were obtained by ultracentrifugation. Proteins in the ghosts and Triton shells were separated on 8% SDS gels followed by staining with Coomassie brilliant blue. The bands indicated by an open arrowhead contain serum albumin in invagination-derived vesicles.¹⁴ (E) Filtration-induced hemolysis generated fragmented vesicles in the filtrates. RBCs from the animals were pressurized on the membrane filter and the vesicles in the filtrate were collected by ultracentrifugation and washed once. Proteins in the vesicles were directly solubilized in the sample buffer for SDS-PAGE, and half the volume of the sample was loaded on 8% SDS gels followed by staining with Coomassie blue. Note that the vesicles from type 1 RBCs were dissolved in 5× the volume of buffer and one-tenth of the solubilized sample, that is equivalent to the volume of other samples, was applied to the well. α- and β-spectrin are apparent only in the vesicles from type 1 band 3–deficient RBCs (arrowheads). Migrating positions of α- and β-spectrin, band 3, 4.1R, 4.2, actin, and globins in ghost membranes, including those of size marker proteins found in kDa, are indicated in panels D-E. ns, not significant.

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